相关申请的交叉引用Cross References to Related Applications
本申请要求保护2008年12月19日提交的美国临时专利申请号61/139,218的优先权权益，该申请的公开内容通过引用以其整体明确结合到本文中。This application claims the benefit of priority to US Provisional Patent Application No. 61/139,218, filed December 19, 2008, the disclosure of which is expressly incorporated herein by reference in its entirety.
背景发明background invention
胰岛素是一种肽激素，由二链异二聚体组成，所述异二聚体以生物合成方式由低效能单链胰岛素原前体通过酶加工获得。人胰岛素由通过二硫键结合在一起的两条肽链(“A链”(SEQ ID NO：1)和“B链”(SEQ ID NO：2))组成，共有51个氨基酸。B链的C端区和A链的2个末端区以三维结构缔合装配成以高亲和力结合胰岛素受体的部位。Insulin is a peptide hormone consisting of a two-chain heterodimer obtained biosynthetically from the enzymatic processing of a low potency single-chain proinsulin precursor. Human insulin consists of two peptide chains ("A chain" (SEQ ID NO: 1) and "B chain" (SEQ ID NO: 2)) held together by disulfide bonds, with a total of 51 amino acids. The C-terminal region of the B chain and the two terminal regions of the A chain are associated in a three-dimensional structure to form a site that binds to the insulin receptor with high affinity.
对于几乎所有形式的糖尿病，胰岛素都具有无以伦比的降低葡萄糖的能力。遗憾的是，其药理学并不是葡萄糖敏感性的，因此它能够产生可导致危及生命的低血糖症的过度作用。不稳定的药理学作用是胰岛素疗法的标志，使得要使血糖正常化而又不发生低血糖症极为困难。此外，天然胰岛素的作用持续时间短，而且天然胰岛素需要修饰使之适用于基础葡萄糖的控制。已确立的延迟胰岛素起效的方法包括降低溶解度和白蛋白结合。Insulin has an unmatched ability to lower glucose for nearly all forms of diabetes. Unfortunately, its pharmacology is not glucose sensitive, so it can produce excessive effects that can lead to life-threatening hypoglycemia. Instability in pharmacology is a hallmark of insulin therapy, making normalization of blood glucose without hypoglycemia extremely difficult. In addition, native insulin has a short duration of action and requires modifications to make it suitable for basal glucose control. Established methods of delaying the onset of insulin action include reducing solubility and albumin binding.
例如，已制备了两种市售的胰岛素衍生物以提供较长的作用特征。更具体地讲，制备了胰岛素衍生物[GlyA21，ArgB31，ArgB32]胰岛素，以使胰岛素的pI由5.4变为6.7，导致该肽在生理pH下沉淀，由此迟缓吸收和作用时间(参见Bolli等，Diabetologia 1999，42，1151-1167)。然而，这种胰岛素衍生物的IGF-1亲和力升高，导致增殖作用增强和肿瘤发生的可能性增加。另一种市售的胰岛素衍生物是[LysB29-十四烷酰基，des(B30)]胰岛素，其中LysB29用C14脂肪酸酰化(Mayer等，Peptide Science，88，5，687-713)。脂肪酸链的存在促进肽与血清白蛋白的结合，导致血浆半寿期延长。然而，这种衍生物具有体内效能降低的缺点。另外，两种胰岛素衍生物的生物作用在一名患者与另一患者间具有差异。For example, two commercially available insulin derivatives have been prepared to provide a longer profile of action. More specifically, insulin derivatives [GlyA21, ArgB31, ArgB32] insulin were prepared to change the pI of insulin from 5.4 to 6.7, causing the peptide to precipitate at physiological pH, thereby slowing absorption and duration of action (see Bolli et al. , Diabetologia 1999, 42, 1151-1167). However, this insulin derivative has an increased affinity for IGF-1, leading to enhanced proliferative effects and increased potential for tumorigenesis. Another commercially available insulin derivative is [LysB29-tetradecanoyl, des(B30)] insulin, in which LysB29 is acylated with aC14 fatty acid (Mayer et al., Peptide Science, 88, 5, 687-713). The presence of fatty acid chains facilitates binding of the peptide to serum albumin, resulting in increased plasma half-life. However, such derivatives have the disadvantage of reduced potency in vivo. In addition, the biological effects of the two insulin derivatives varied from one patient to another.
前药化学法为在从给药部位清除并在血浆中以高度限定的浓度平衡后精确控制胰岛素作用的开始和持续时间提供了机会。与现有的长效胰岛素类似物和制剂相比，该方法的主要优点是胰岛素贮库不是其中进行注射的皮下脂肪组织，而是血液区室。这就排除了现有技术延迟胰岛素衍生物起效所遇到的吸收变化性。这还使通过皮下注射以外的途径给予该肽激素成为可能。Prodrug chemistry offers the opportunity to precisely control the onset and duration of insulin action after clearance from the site of administration and equilibration in plasma at highly defined concentrations. The main advantage of this approach over existing long-acting insulin analogs and formulations is that the insulin depot is not the subcutaneous adipose tissue where the injection takes place, but the blood compartment. This precludes the variability in absorption encountered with prior art delays in the onset of action of insulin derivatives. This also makes it possible to administer the peptide hormone by routes other than subcutaneous injection.
胰岛素与其受体结合将引起生物刺激，但也将通过胰岛素肽的酶促降解而启动胰岛素诱导的药理性质的后续钝化。使用胰岛素前药衍生物的额外优势是这类方法还根据抑制前药被相应受体识别的策略，来延长胰岛素的生物半寿期。尽管这些优势与前药衍生物相关，但是制备这类前药的复杂特性至今一直妨碍有效胰岛素前药衍生物的制备。为了开发成功的前药激素，需要活性部位结构地址(structural address)，其可构成用于前药结构元件可逆附着的基础。该结构地址需提供两个关键特征；(1)选择性化学修饰的潜力，和(2)在脱去前药结构元件时以天然形式提供高度活性的能力。将本文公开的胰岛素前药以化学法转化为可被受体识别的结构，其中该化学转变的速度将决定体内生物作用的开始和持续时间。本申请公开的前药化学法有赖于分子内的化学反应，该反应不依赖其它化学添加剂或者酶或酶抑制剂。Binding of insulin to its receptor will cause biostimulation, but will also initiate the subsequent inactivation of insulin-induced pharmacological properties by enzymatic degradation of the insulin peptide. An additional advantage of using insulin prodrug derivatives is that such approaches also prolong the biological half-life of insulin based on a strategy of inhibiting the recognition of the prodrug by the corresponding receptor. Despite these advantages associated with prodrug derivatives, the complex nature of preparing such prodrugs has so far prevented the preparation of effective insulin prodrug derivatives. In order to develop successful prodrug hormones, active site structural addresses are required, which may form the basis for reversible attachment of prodrug structural elements. This structural address needs to provide two key features; (1) the potential for selective chemical modification, and (2) the ability to provide high activity in the native form upon removal of the prodrug structural element. The insulin prodrugs disclosed herein are chemically converted to a receptor-recognizable structure, wherein the speed of this chemical conversion will determine the onset and duration of biological action in vivo. The prodrug chemistry disclosed in this application relies on intramolecular chemical reactions that do not rely on other chemical additives or enzymes or enzyme inhibitors.
理想的前药在生理条件(例如pH 7.2和37℃)下应可溶于水，并且以粉末形式长期保存时应保持稳定。与母体药物相比，前药还应是免疫沉默的，并具有低活性。前药的活性通常不超过母体药物活性的10％，在一个实施方案中，与母体药物相比，前药的活性小于10％、小于5％、约1％或小于1％。此外，前药当注射到体内时，应在规定时段内定量转化为活性药物。申请人首次公开了符合这些目标中的每一项的胰岛素前药类似物。An ideal prodrug should be soluble in water under physiological conditions (e.g., pH 7.2 and 37 °C) and should remain stable in powder form for long-term storage. Prodrugs should also be immunosilent and have low activity compared to the parent drug. Prodrugs typically have no more than 10% of the activity of the parent drug, and in one embodiment, the prodrug has less than 10%, less than 5%, about 1%, or less than 1% of the activity of the parent drug. Furthermore, the prodrug, when injected into the body, should be quantitatively converted to the active drug within a defined period of time. Applicants are the first to disclose insulin prodrug analogs that meet each of these objectives.
发明概述Summary of the invention
基于肽的药物是十分有效的药物，其作用持续时间相对较短，治疗指数可变。本公开内容涉及胰岛素前药，其中将前药衍生物设计成起效延迟，而且药物半寿期延长。起效延迟是有利的，因为允许前药在其活化前系统分布。因此，给予前药排除了在给药时由峰值活性引起的并发症，并提高母体药物的治疗指数。Peptide-based drugs are very potent drugs with a relatively short duration of action and variable therapeutic index. The present disclosure relates to insulin prodrugs wherein prodrug derivatives are designed to have a delayed onset of action and an increased drug half-life. A delayed onset of action is advantageous because it allows for systemic distribution of the prodrug prior to its activation. Administration of the prodrug thus precludes complications arising from peak activity upon dosing and increases the therapeutic index of the parent drug.
根据一个实施方案，将二肽通过酰胺键与胰岛素肽的活性部位共价连接来制备胰岛素的前药衍生物。在一个实施方案中，二肽在干扰胰岛素与胰岛素受体和IGF-1受体相互作用的能力的位置上与胰岛素肽共价结合。随后在生理条件下且缺乏酶活性时，通过导致二酮哌嗪或二酮吗啉形成的分子内反应除去二肽，恢复多肽的完整活性。According to one embodiment, a prodrug derivative of insulin is prepared by covalently linking a dipeptide to the active site of the insulin peptide via an amide bond. In one embodiment, the dipeptide is covalently bound to the insulin peptide at a position that interferes with the ability of insulin to interact with the insulin receptor and the IGF-1 receptor. Subsequent removal of the dipeptide under physiological conditions and in the absence of enzymatic activity restores full activity of the polypeptide by an intramolecular reaction leading to the formation of diketopiperazine or diketomorpholine.
在一个实施方案中，提供具有U-O-胰岛素的通用结构的胰岛素前药，其中U为氨基酸或羟酸，O为通过在U-O和胰岛素肽的胺之间形成酰胺键而与胰岛素肽连接的N-烷基化氨基酸。在一个实施方案中，U-O二肽在A链或B链各自的N端或与A链或B链各自的A19、B16或B25位对应的氨基酸侧链上通过酰胺键结合。在一个实施方案中，选择U-O的结构，其中在生理条件下，在PBS中，在约1小时-约720小时内U-O自胰岛素肽的化学裂解完成至少约90％。在一个实施方案中，在生理条件下，在PBS中，U-O自胰岛素肽的化学裂解半寿期(t1/2)为至少约1小时-约1周。In one embodiment, there is provided an insulin prodrug having the general structure of UO-insulin, wherein U is an amino acid or a hydroxyacid, O is an N- Alkylated amino acids. In one embodiment, the UO dipeptide is bound via an amide bond at the N-terminus of the A chain or the B chain respectively or on the amino acid side chain corresponding to the A19, B16 or B25 positions of the A chain or the B chain respectively. In one embodiment, the structure of UO is selected wherein chemical cleavage of UO from insulin peptide is at least about 90% complete within about 1 hour to about 720 hours in PBS under physiological conditions. In one embodiment, the chemical cleavage half-life (t1/2 ) of UO from insulin peptide is at least about 1 hour to about 1 week in PBS under physiological conditions.
在一个实施方案中，选择U和O以抑制存在于哺乳动物血清中的酶从胰岛素肽上酶促切割U-O二肽。在一个实施方案中，选择U和/或O，使得在生理条件下，在PBS中，从胰岛素肽对U-O的裂解半寿期不大于在包含DPP-IV蛋白酶的溶液中从胰岛素肽对U-O的裂解半寿期的两倍(即与不存在DPP-IV蛋白酶的相同条件相比，在该酶存在和生理条件下从胰岛素前药中裂解U-O不会以超过2倍的速率发生)。在一个实施方案中，U、O或U-O与之连接的胰岛素肽的氨基酸为非编码氨基酸。在一个实施方案中，U和/或O为呈D立体异构体构型的氨基酸。在一些示例性的实施方案中，U为呈D立体异构体构型的氨基酸，而O为L立体异构体构型的氨基酸。在一些示例性的实施方案中，U为呈L立体异构体构型的氨基酸，而O为呈D立体异构体构型的氨基酸。在一些示例性的实施方案中，U为呈D立体异构体构型的氨基酸，而O为呈D立体异构体构型的氨基酸。在一个实施方案中，O为N-烷基化氨基酸，但不是脯氨酸。In one embodiment, U and O are selected to inhibit the enzymatic cleavage of the U-O dipeptide from the insulin peptide by enzymes present in mammalian serum. In one embodiment, U and/or O are selected such that under physiological conditions, the cleavage half-life of U-O from the insulin peptide in PBS is not greater than that of U-O from the insulin peptide in a solution containing DPP-IV protease. Twice the cleavage half-life (i.e., cleavage of U-O from the insulin prodrug does not occur at more than a factor of two in the presence of the enzyme and under physiological conditions compared to the same conditions in the absence of DPP-IV protease). In one embodiment, the amino acid of the insulin peptide to which the U, O, or U-O is linked is a non-coded amino acid. In one embodiment, U and/or O are amino acids in the D stereoisomer configuration. In some exemplary embodiments, U is an amino acid in the D stereoisomer configuration and O is an amino acid in the L stereoisomer configuration. In some exemplary embodiments, U is an amino acid in the L stereoisomer configuration and O is an amino acid in the D stereoisomer configuration. In some exemplary embodiments, U is an amino acid in the D stereoisomer configuration and O is an amino acid in the D stereoisomer configuration. In one embodiment, O is an N-alkylated amino acid, but is not proline.
在一个实施方案中，二肽前药元件包含具有以下式I通用结构的化合物：In one embodiment, the dipeptide prodrug element comprises a compound having the general structure of Formula I below:
其中in
R1、R2、R4和R8独立选自H、C1-C18烷基、C2-C18烯基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基(heterocyclic))、(C0-C4烷基)(C6-C10芳基)R7、(C1-C4烷基)(C3-C9杂芳基)和C1-C12烷基(W)C1-C12烷基，其中W为选自N、S和O的杂原子，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基或芳基；或者R4和R8与它们所连接的原子一起形成C3-C6环烷基；R1 , R2 , R4 and R8 are independently selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C1 -C18 alkyl)OH, (C1 -C18 alkane base) SH, (C2 -C3 alkyl) SCH3 , (C1 -C4 alkyl) CONH2 , (C1 -C4 alkyl) COOH, (C1 -C4 alkyl) NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclic group (heterocyclic)), (C0 -C4 alkyl) (C6 -C10 aryl) R7 , (C1 -C4 alkyl) (C3 -C9 heteroaryl group) and C1 -C12 alkyl (W) C1 -C12 alkyl, wherein W is a heteroatom selected from N, S and O, or R1 and R2 together with the atoms to which they are attached form C3 -C12 cycloalkyl or aryl; or R4 and R8 form C3 -C6 cycloalkyl together with the atoms they are connected to;
R3选自C1-C18烷基、(C1-C18烷基)OH、(C1-C18烷基)NH2、(C1-C18烷基)SH、(C0-C4烷基)(C3-C6)环烷基、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和(C1-C4烷基)(C3-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成4、5或6元杂环；R3 is selected from C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C18 alkyl) NH2 , (C1 -C18 alkyl) SH, (C0 - C4 alkyl) (C3 -C6 ) cycloalkyl, (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and (C1 -C4 alkyl) (C3 -C9 heteroaryl), or R4 and R3 form a 4, 5 or 6 membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R2与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R2 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自H和OH。R7 is selected from H and OH.
在一个实施方案中，二肽前药元件包含具有以下式I通用结构的化合物：In one embodiment, the dipeptide prodrug element comprises a compound having the general structure of Formula I below:
其中in
R1、R2、R4和R8独立选自H、C1-C18烷基、C2-C18烯基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7、(C1-C4烷基)(C3-C9杂芳基)和C1-C12烷基(W1)C1-C12烷基，其中W1为选自N、S和O的杂原子，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基；或者R4和R8与它们所连接的原子一起形成C3-C6环烷基；R1 , R2 , R4 and R8 are independently selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C1 -C18 alkyl)OH, (C1 -C18 alkane base) SH, (C2 -C3 alkyl) SCH3 , (C1 -C4 alkyl) CONH2 , (C1 -C4 alkyl) COOH, (C1 -C4 alkyl) NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 , (C1 -C4 alkyl) (C3 -C9 heteroaryl) and C1 -C12 alkyl (W1 )C1 -C12 alkyl, wherein W1 is a heteroatom selected from N, S and O, or R1 and R2 together with the atoms to which they are attached form C3 -C12 cycloalkyl; or R4 and R8 form C3 -C6 cycloalkyl together with the atoms they are connected to;
R3选自C1-C18烷基、(C1-C18烷基)OH、(C1-C18烷基)NH2、(C1-C18烷基)SH、(C0-C4烷基)(C3-C6)环烷基、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和(C1-C4烷基)(C3-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成4、5或6元杂环；R3 is selected from C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C18 alkyl) NH2 , (C1 -C18 alkyl) SH, (C0 - C4 alkyl) (C3 -C6 ) cycloalkyl, (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and (C1 -C4 alkyl) (C3 -C9 heteroaryl), or R4 and R3 form a 4, 5 or 6 membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
在一个实施方案中，二肽延伸物包含以下通用结构的化合物：In one embodiment, the dipeptide extension comprises a compound of the general structure:
其中R1选自H和C1-C8烷基；和wherein R is selected from H and C1-C 8alkyl ; and
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C8环烷基环；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 and CH2 (C5 -C9 heteroaryl), or R1 and R2 together with the atoms they are connected to form a C3 -C8 cycloalkyl ring;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, or R6 and R2together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring; and
R7选自H和OH。在一个实施方案中，R3为C1-C8烷基，R4选自H、C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成5或6元杂环。R7 is selected from H and OH. In one embodiment, R3 is C1 -C8 alkyl, R4 is selected from H, C1 -C8 alkyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl )SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and CH2 (C5 -C9 heteroaryl), or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms to which they are attached.
根据一个实施方案，提供包含A链和B链的胰岛素前药类似物，其中A链包含序列Z-GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)，B链包含序列J-X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)。A链和B链式中的指称Z和J独立地为H(形成N端胺)或包含以下通用结构的二肽：According to one embodiment, there is provided an insulin prodrug analogue comprising a chain A comprising the sequence Z-GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) and a chain B comprising the sequence JX14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14). Designations Z and J in chain A and chain B are independently H (forming an N-terminal amine) or a dipeptide comprising the following general structure:
其中 in
X14为将“J”元件与X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)序列的N端连接的键，或者X14表示将“J”元件与X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)序列的N端连接的选自以下的1-4个氨基酸的序列：FVNQ(SEQ ID NO：11)、VNQ、NQ和Q。X14 is the bond connecting the "J" element to the N-terminus of the sequence X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14), or X14 represents the linkage of the "J" element to X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14) sequence N-terminally linked to a sequence of 1-4 amino acids selected from the group consisting of FVNQ (SEQ ID NO: 11 ), VNQ, NQ and Q.
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NH2、NHR10和OCH3，其中R10为包含以下通用结构的二肽：wherein X is selected from OH, NH2 , NHR10 and OCH3 , wherein R10 is a dipeptide comprising the following general structure:
X3选自天冬酰胺、鸟氨酸、甘氨酸、丙氨酸、苏氨酸和丝氨酸；X is selected from asparagine,ornithine , glycine, alanine, threonine and serine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X7为以下通用结构的氨基酸X7 is an amino acid of the following general structure
其中X12选自OH、NH2、NHR11和OCH3，其中R11为包含以下通用结构的二肽：Wherein X12 is selected from OH, NH2 , NHR11 and OCH3 , wherein R11 is a dipeptide comprising the following general structure:
X8为以下通用结构的氨基酸X8 is an amino acid of the following general structure
其中X13选自H、OH、NH2、NHR12和OCH3，其中R12为包含以下通用结构的二肽：Wherein X13 is selected from H, OH, NH2 , NHR12 and OCH3 , wherein R12 is a dipeptide comprising the following general structure:
其中R1选自H和C1-C8烷基；Wherein R1 is selected from H and C1 -C8 alkyl;
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 and CH2 (C5 -C9 heteroaryl);
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C3 -C6 ) cycloalkyl, or R4 and R3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclic ring;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, or R6 and R2together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring; and
R7选自H和OH，前提条件是X、X12、X13、J和Z中的一个并且只有一个包含以下通用结构的二肽：R7 is selected from H and OH with the proviso that one of X,X12 ,X13 , J and Z and only one dipeptide comprising the following general structure:
在一个实施方案中，如果J或Z包含式I的二肽，且R4和R3与它们所连接的原子一起形成4、5或6元杂环，则R1和R2两者都不为氢。 In one embodiment, if J or Z comprises a dipeptide of formula I, and R andR together with the atoms to which they are attached form a4 ,5 or6 membered heterocyclic ring, neither R nor R for hydrogen.
根据一个实施方案，存在于Z、J、R10、R11或R12上的二肽包含具有下式I通用结构的化合物：According to one embodiment, the dipeptide present on Z, J, R10 , R11 or R12 comprises a compound having the general structure of formula I below:
其中in
R1、R2、R4和R8独立选自H、C1-C18烷基、C2-C18烯基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7、(C1-C4烷基)(C3-C9杂芳基)和C1-C12烷基(W1)C1-C12烷基，其中W1为选自N、S和O的杂原子，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基；或者R4和R8与它们所连接的原子一起形成C3-C6环烷基；R1 , R2 , R4 and R8 are independently selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C1 -C18 alkyl)OH, (C1 -C18 alkane base) SH, (C2 -C3 alkyl) SCH3 , (C1 -C4 alkyl) CONH2 , (C1 -C4 alkyl) COOH, (C1 -C4 alkyl) NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 , (C1 -C4 alkyl) (C3 -C9 heteroaryl) and C1 -C12 alkyl (W1 )C1 -C12 alkyl, wherein W1 is a heteroatom selected from N, S and O, or R1 and R2 together with the atoms to which they are attached form C3 -C12 cycloalkyl; or R4 and R8 form C3 -C6 cycloalkyl together with the atoms they are connected to;
R3选自C1-C18烷基、(C1-C18烷基)OH、(C1-C18烷基)NH2、(C1-C18烷基)SH、(C0-C4烷基)(C3-C6)环烷基、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和(C1-C4烷基)(C3-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成4、5或6元杂环；R3 is selected from C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C18 alkyl) NH2 , (C1 -C18 alkyl) SH, (C0 - C4 alkyl) (C3 -C6 ) cycloalkyl, (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and (C1 -C4 alkyl) (C3 -C9 heteroaryl), or R4 and R3 form a 4, 5 or 6 membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
根据一个实施方案，存在于Z、J、R10、R11或R12上的二肽包含具有下式I通用结构的化合物：According to one embodiment, the dipeptide present on Z, J, R10 , R11 or R12 comprises a compound having the general structure of formula I below:
其中in
R1和R8独立地为H或C1-C8烷基；R1 and R8 are independently H or C1 -C8 alkyl;
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C3-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2 +) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclic radical), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and CH2 (C3 -C9 heteroaryl), or R1 and R2 together with the atoms to which they are attached Formation of a C3 -C12 cycloalkyl group;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R5为NHR6；R5 is NHR6 ;
R6为H或C1-C8烷基；和R6 is H or C1 -C8 alkyl; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
根据一个实施方案，提供胰岛素类似物，其中胰岛素肽的A链包含序列Z-GIVEQCCTSICSLYQLENX2CN(SEQ ID NO：6)，B链包含选自以下的序列：HLCGSHLVEALYLVCGERGFF(SEQ ID NO：7)、FVNQHLCGSHLVEALYLVCGERGFFYTPKT(SEQ ID NO：8)和FVNQHLCGSHLVEALYLVCGERGFFYTKPT(SEQ ID NO：9)，其中Z为H或包含以下通用结构的二肽：According to one embodiment, there is provided an insulin analogue wherein the A chain of the insulin peptide comprises the sequence Z-GIVEQCCTSICSLYQLENX2 CN (SEQ ID NO: 6) and the B chain comprises a sequence selected from the group consisting of: HLCGSHLVEALYLVCGERGFF (SEQ ID NO: 7), FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 8) and FVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 9), wherein Z is H or a dipeptide comprising the following general structure:
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中R10为H或包含以下通用结构的二肽：wherein R is H or adipeptide comprising the following general structure:
前提条件是Z和R10不同时为H，且不同时为包含以下通用结构的二肽：The prerequisite is that Z and R10 are not both H and not simultaneously a dipeptide comprising the following general structure:
根据一个实施方案，提供单链胰岛素前药类似物。在这个实施方案中，人胰岛素B链或其功能类似物的羧基端与人胰岛素A链或其功能类似物的N端共价连接，其中具有以下通用结构的二肽前药部分在肽的N端或者在与A链或B链各自的A19、B16或B25位对应的氨基酸侧链上通过酰胺键共价结合：According to one embodiment, a single chain insulin prodrug analog is provided. In this embodiment, the carboxy-terminus of the B chain of human insulin or a functional analog thereof is covalently linked to the N-terminus of the A chain of human insulin or a functional analog thereof, wherein a dipeptide prodrug moiety having the following general structure is at the N-terminus of the peptide or on the amino acid side chain corresponding to the A19, B16 or B25 positions of the A chain or the B chain respectively via an amide bond:
在一个实施方案中，B链通过4-12个或4-8个氨基酸的肽接头与A链连接。 In one embodiment, the B chain is linked to the A chain by a peptide linker of 4-12 or 4-8 amino acids.
在另一个实施方案中，将胰岛素前药类似物的溶解度通过将亲水部分与肽共价连接来提高。在一个实施方案中，亲水部分与B链的N端氨基酸或与SEQ ID NO：9的28位氨基酸或SEQ ID NO：8的29位氨基酸连接。在一个实施方案中，亲水部分为分子量范围为约500-约40,000道尔顿的聚乙二醇(PEG)链。在一个实施方案中，聚乙二醇链的分子量范围选自约500-约5,000道尔顿。在另一个实施方案中，聚乙二醇链的分子量为约10,000-约20,000道尔顿。In another embodiment, the solubility of the insulin prodrug analog is enhanced by covalently attaching a hydrophilic moiety to the peptide. In one embodiment, the hydrophilic moiety is linked to the N-terminal amino acid of the B chain or to amino acid 28 of SEQ ID NO:9 or amino acid 29 of SEQ ID NO:8. In one embodiment, the hydrophilic portion is a polyethylene glycol (PEG) chain having a molecular weight ranging from about 500 to about 40,000 Daltons. In one embodiment, the molecular weight of the polyethylene glycol chains is selected from the range of about 500 to about 5,000 Daltons. In another embodiment, the molecular weight of the polyethylene glycol chains is from about 10,000 to about 20,000 Daltons.
酰化或烷基化可增加胰岛素肽在循环中的半寿期。在前药激活时，酰化或烷基化可有利地延迟对胰岛素受体的起效和/或延长对胰岛素受体的作用持续时间。胰岛素类似物可以在与亲水部分所连接氨基酸位置相同的氨基酸位置上酰化或烷基化，或者在不同的氨基酸位置上酰化或烷基化。Acylation or alkylation increases the half-life of insulin peptides in circulation. Upon prodrug activation, acylation or alkylation may advantageously delay the onset and/or prolong the duration of action at the insulin receptor. The insulin analog can be acylated or alkylated at the same amino acid position as the amino acid position to which the hydrophilic moiety is attached, or at a different amino acid position.
根据一个实施方案，提供包含本文公开的任何新的胰岛素前药类似物和药学上可接受的稀释剂、载体或赋形剂的药物组合物，优选所述胰岛素前药类似物的纯度水平为至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％。这类组合物可含有本文公开的A19胰岛素类似物，其浓度为至少0.5mg/ml、1mg/ml、2mg/ml、3mg/ml、4mg/ml、5mg/ml、6mg/ml、7mg/ml、8mg/ml、9mg/ml、10mg/ml、11mg/ml、12mg/ml、13mg/ml、14mg/ml、15mg/ml、16mg/ml、17mg/ml、18mg/ml、19mg/ml、20mg/ml、21mg/ml、22mg/ml、23mg/ml、24mg/ml、25mg/ml或更高。在一个实施方案中，药物组合物包含经过灭菌并任选装入各种包装容器中保存的含水溶液剂。在其它实施方案中，药物组合物包含冻干散剂。药物组合物可进一步作为试剂盒的部分包装，所述试剂盒包括用于将组合物给予患者的一次性装置。容器或试剂盒可贴上在环境室温下保存或在冷冻温度下保存的标签。According to one embodiment, there is provided a pharmaceutical composition comprising any novel insulin prodrug analog disclosed herein and a pharmaceutically acceptable diluent, carrier or excipient, preferably said insulin prodrug analog has a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Such compositions may contain an A19 insulin analog disclosed herein at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml , 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, 20mg /ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml or higher. In one embodiment, the pharmaceutical compositions comprise aqueous solutions which are sterilized and optionally stored in various packaging containers. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may further be packaged as part of a kit comprising a disposable device for administering the composition to a patient. Containers or kits may be labeled for storage at ambient room temperature or storage at refrigerated temperatures.
根据一个实施方案，提供调节胰岛素依赖性患者的血糖水平的改进方法。该方法包括按对控制糖尿病治疗有效的量给予本公开内容的胰岛素前药类似物的步骤。在一个实施方案中，将胰岛素前药类似物用分子量范围为约5,000-约40,000道尔顿的PEG链聚乙二醇化。According to one embodiment, improved methods of regulating blood glucose levels in insulin dependent patients are provided. The method includes the step of administering an insulin prodrug analog of the present disclosure in an amount that is therapeutically effective for controlling diabetes. In one embodiment, the insulin prodrug analog is pegylated with a PEG chain having a molecular weight ranging from about 5,000 to about 40,000 Daltons.
附图简述Brief description of the drawings
图1是制备人胰岛素的两步合成策略的示意图。该方法的详情见实施例1。Figure 1 is a schematic diagram of a two-step synthetic strategy for the preparation of human insulin. See Example 1 for details of this method.
图2是比较合成人胰岛素相对于经纯化的天然胰岛素的胰岛素受体特异性结合的曲线图。图中所提供的数据表明，两种分子具有相似的结合活性。Figure 2 is a graph comparing the insulin receptor specific binding of synthetic human insulin relative to purified native insulin. The data presented in the figure shows that both molecules have similar binding activity.
图3是比较天然胰岛素和A19胰岛素类似物(胰岛素(p-NH2-F)19)的相对胰岛素受体结合的曲线图。图中所提供的数据表明，两种分子具有相似的结合活性。Figure 3 is a graph comparing the relative insulin receptor binding of native insulin and the A19 insulin analog (insulin(p-NH2 -F)19 ). The data presented in the figure shows that both molecules have similar binding activity.
图4是比较天然胰岛素和IGF1(YL)B16B17类似物的相对胰岛素受体结合的曲线图。图中所提供的数据表明，两种分子具有相似的结合活性。Figure 4 is a graph comparing the relative insulin receptor binding of native insulin and IGF1(YL)B16B17 analogs. The data presented in the figure shows that both molecules have similar binding activity.
图5是制备IGF1(YB16LB17)(p-NH2-F)A19类似物(不会被激活)所用的合成流程示意图。Figure 5 is a schematic diagram of the synthetic process used to prepare IGF1(YB16 LB17 )(p-NH2 -F)A19 analogues (which will not be activated).
图6是比较IGF1(YB16LB17)(p-NH2-F)A19和IGF1(YB16LB17)(p-NH2-F)A19的二肽延伸形式的相对胰岛素受体结合的曲线图，其中二肽AiBAla在A19位上结合(即IGF1(YB16LB17)A19-AiBAla)。Figure 6 is a graph comparing the relative insulin receptor binding of IGF1(YB16 LB17 )(p-NH2 -F)A19 and dipeptide extended forms of IGF1(YB16 LB17 )(p-NH2 -F)A19 Figure, wherein the dipeptide AiBAla binds at position A19 (ie IGF1(YB16 LB17 )A19 -AiBAla).
图7A-7C给出按照本公开内容制备的二聚体的活性。图7A显示IGF-1单链二聚体的结构，该二聚体包含通过B链氨基端的侧链间的二硫键连接在一起的2条单链IGFB16B17衍生肽(IGF-1B链[C0H5Y16L17O22]-A链[O9，14，15N18，21]；SEQ ID NO：68)。图7B为显示胰岛素、IGF-1、单链IGFB16B17衍生肽二聚体和二链IGFB16B17衍生肽二聚体的相对胰岛素受体结合的曲线图。图7C为显示胰岛素、IGF-1和二链IGFB16B17衍生肽二聚体诱导胰岛素受体磷酸化的相对活性的曲线图。Figures 7A-7C show the activity of dimers prepared according to the present disclosure. Figure 7A shows the structure of an IGF-1 single-chain dimer comprising two single-chain IGFB16B17 -derived peptides (IGF-1B chain [C0 H5 Y16 L17 O22 ]-A chain [O9,14,15 N18,21 ]; SEQ ID NO:68). Figure 7B is a graph showing the relative insulin receptor binding of insulin, IGF-1, single-chain IGFB16B17- derived peptide dimers, and two-chain IGFB16B17- derived peptide dimers. Figure 7C is a graph showing the relative activities of insulin, IGF-1 and two-chain IGFB16B17- derived peptide dimers to induce insulin receptor phosphorylation.
图8A-8C显示前药形式的IGFB16B17衍生肽(IGF1A(Ala)6，7，11，20酰胺的(pNH2-F)19上的Aib-Pro)的降解。将二肽在PBS(pH 7.4)中于37℃保温预定长度的时间。在开始保温后20分钟(图8A)、81分钟(图8B)和120分钟(图8C)取出等分量，用0.1％TFA淬灭，然后用分析型HPLC测定。用LC-MS鉴定a峰(IGF1A(Ala)6，7，11，20(pNH2-F)1酰胺)和b峰(IGF1A(Ala)6，7，11，20(Aib-Pro-pNH-F)19酰胺)，并通过峰面积积分来定量。数据表明IGF1A(Ala)6，7，11，20(Aib-Pro-pNH-F)19酰胺随时间自发非酶促地转化成IGF1A(Ala)6，7，11，20(pNH2-F)1酰胺。Figures 8A-8C show the degradation of the IGFB16B17- derived peptide (Aib-Pro on (pNH2- F)19 of the IGF1A(Ala)6,7,11,20 amide) in prodrug form. The dipeptides were incubated in PBS (pH 7.4) at 37°C for a predetermined length of time. Aliquots were taken at 20 minutes (FIG. 8A), 81 minutes (FIG. 8B) and 120 minutes (FIG. 8C) after the start of the incubation, quenched with 0.1% TFA, and assayed by analytical HPLC. Using LC-MS to identify peak a (IGF1A(Ala)6 , 7, 11, 20 (pNH2 -F)1 amide) and peak b (IGF1A(Ala) 6, 7, 11,20 (Aib-Pro-pNH- F)19 amide), and quantified by peak area integration. Data indicate spontaneous non-enzymatic conversion of IGF1A(Ala)6,7,11,20 (Aib-Pro-pNH-F)19amide to IGF1A(Ala)6,7,11,20 (pNH2- F) over time1 amide.
图9A和图9B为表示前药Aib，dPro-IGF1YL(通过A19 4-氨基Phe连接的二肽)的体外活性的曲线图。图9A是比较在PBS中保温的天然胰岛素(于4℃下在1小时时测定)和A19IGF前药类似物(Aib，dPro-IGF1YL)随时间变化(0小时、2.5小时和10.6小时)的相对胰岛素受体结合的曲线图。图9B是比较在20％血浆/PBS中保温的天然胰岛素(于4℃下在1.5小时时测定)和A19 IGF前药类似物(Aib，dPro-IGF1YL)随时间变化(0小时、1.5小时和24.8小时)的相对胰岛素受体结合的曲线图。图中所提供的数据表明，随着前药形式转化成活性IGF1YL肽，从A19IGF前药类似物样品恢复的活性渐增。Figures 9A and 9B are graphs showing the in vitro activity of the prodrug Aib, dPro-IGF1YL (dipeptide linked through A19 4-aminoPhe). Figure 9A is a comparison of native insulin (measured at 4°C at 1 hour) and A19IGF prodrug analogs (Aib, dPro-IGF1YL) incubated in PBS over time (0 hours, 2.5 hours and 10.6 hours). Graph of insulin receptor binding. Figure 9B is a comparison of natural insulin (measured at 1.5 hours at 4°C) and A19 IGF prodrug analogs (Aib, dPro-IGF1YL) incubated in 20% plasma/PBS over time (0 hours, 1.5 hours and 24.8 hours) relative insulin receptor binding curve. The data presented in the figure demonstrates that the recovery of activity from the A19IGF prodrug analog samples is progressive as the prodrug form is converted to the active IGF1YL peptide.
图10A和图10B为描述前药dK，(N-异丁基G)-IGF1YL(通过A194-氨基Phe连接的二肽)的体外活性的曲线图。图10A是比较在PBS中保温的天然胰岛素(在4℃下于1小时时测定)和A19 IGF前药类似物(IGF1YL:dK，(N-异丁基G)随时间变化(0小时、5小时和52小时)的相对胰岛素受体结合的曲线图。图10B是比较在20％血浆/PBS中保温的天然胰岛素(于4℃下在1.5小时时测定)和A19IGF前药类似物(IGF1YL:dK，(N-异丁基G)随时间变化(0小时、3.6小时和24.8小时)的相对胰岛素受体结合的曲线图。图中所提供的数据表明，随着前药形式转化成活性IGF1YL肽，从A19IGF前药类似物样品恢复的活性渐增。Figures 10A and 10B are graphs depicting the in vitro activity of the prodrug dK, (N-isobutylG)-IGF1YL, a dipeptide linked through A194-aminoPhe. Figure 10A is a comparison of natural insulin (measured at 4°C at 1 hour) and A19 IGF prodrug analogs (IGF1YL:dK, (N-isobutyl G) over time (0 hours, 5 hours) incubated in PBS. hours and 52 hours) of the relative insulin receptor binding curve. Figure 10B is a comparison of native insulin (measured at 1.5 hours at 4° C.) and A19IGF prodrug analogs (IGF1YL: A plot of dK, (N-isobutyl G) relative to insulin receptor binding over time (0 hours, 3.6 hours and 24.8 hours). The data presented in the figure shows that as the prodrug form is converted to active IGF1YL Peptides with increasing activity recovered from A19IGF prodrug analog samples.
图11A和图11B为描述前药dK(e-乙酰基)，Sar)-IGF1YL(通过A19 4-氨基Phe连接的二肽)的体外活性的曲线图。图11A是比较在PBS中保温的天然胰岛素(于4℃下在1小时时测定)和A19 IGF前药类似物(IGF1YL:dK(e-乙酰基)，Sar)随时间变化(0小时、7.2小时和91.6小时)的相对胰岛素受体结合的曲线图。图11B是比较在20％血浆/PBS中保温的天然胰岛素(于4℃在1.5小时时测定)和A19 IGF前药类似物(IGF1YL:dK(e-乙酰基)，Sar)随时间变化(0小时、9小时和95小时)的相对胰岛素受体结合的曲线图。图中所提供的数据表明，随着前药形式转化成活性IGF1YL肽，从A19 IGF前药类似物样品恢复的活性渐增。11A and 11B are graphs depicting the in vitro activity of the prodrug dK(e-acetyl),Sar)-IGF1YL, a dipeptide linked through A19 4-aminoPhe. Figure 11A is a comparison of natural insulin (measured at 4°C at 1 hour) and A19 IGF prodrug analogs (IGF1YL: dK (e-acetyl), Sar) over time (0 hours, 7.2 hours) incubated in PBS. Hours and 91.6 hours) relative insulin receptor binding curves. Figure 11B is a comparison of natural insulin (measured at 4°C at 1.5 hours) and A19 IGF prodrug analogs (IGF1YL: dK(e-acetyl), Sar) over time (0) incubated in 20% plasma/PBS Hours, 9 hours and 95 hours) of the relative insulin receptor binding curves. The data presented in the figure demonstrates that the recovery of activity from the A19 IGF prodrug analog samples is progressive as the prodrug form is converted to the active IGF1YL peptide.
发明详述Detailed description of the invention
定义definition
在描述和要求保护本发明时，根据下文给出的定义使用下列术语。In describing and claiming the present invention, the following terminology is used in accordance with the definitions given hereinafter.
将本文使用的术语“前药”定义为在显示其药理作用之前经历化学修饰的任何化合物。The term "prodrug" as used herein is defined as any compound that undergoes chemical modification prior to exhibiting its pharmacological effects.
“生物活性多肽”是指能够在体外和/或体内施加生物作用的多肽。A "biologically active polypeptide" refers to a polypeptide capable of exerting a biological effect in vitro and/or in vivo.
本文使用的术语“氨基酸”包括既含氨基官能团又含羧基官能团的任何分子，其中氨基和羧酸基连接至同一碳(α碳)上。α碳任选可具有一个或两个其它的有机取代基。未说明其立体化学的情况下对氨基酸的命名意在包括L型或D型氨基酸或外消旋混合物。然而，在氨基酸通过其三字母码命名并包括上标数字的情况下，通过在三字母码和上标数字之前加上小写字母d来指定D型氨基酸(例如dLys-1)，其中缺乏小写字母d的命名(例如Lys-1)意在说明天然L型氨基酸。在该命名法中，加上上标数字表明氨基酸在IGF肽序列中的位置，其中位于IGF序列内的氨基酸用从N端起连续编号的正的上标数字标示。在N端或通过侧链与IGF肽连接的其它氨基酸从0开始编号，并随着其更远离IGF序列，以负整数值递增。例如，与IGF的N端连接的二肽前药内的氨基酸位置标为aa-1-aa0-IGF，其中aa0表示二肽的羧基端氨基酸，aa-1表示二肽的氨基端氨基酸。The term "amino acid" as used herein includes any molecule containing both amino and carboxyl functional groups, wherein the amino and carboxylic acid groups are attached to the same carbon (alpha carbon). The alpha carbon optionally can bear one or two other organic substituents. The designation of an amino acid without specifying its stereochemistry is intended to include the L- or D-form of the amino acid or the racemic mixture. However, where an amino acid is named by its three-letter code and includes a superscript number, the D-type amino acid (e.g. dLys-1 ), which lacks a lowercase letter, is designated by preceding the three-letter code and superscript number with a lowercase d The nomenclature of d (eg Lys-1 ) is intended to illustrate the natural L-form amino acid. In this nomenclature, the addition of a superscript number indicates the position of the amino acid in the IGF peptide sequence, wherein amino acids located within the IGF sequence are indicated by positive superscript numbers numbered consecutively from the N-terminus. Other amino acids linked to the IGF peptide at the N-terminus or through side chains are numbered starting from 0 and increasing with negative integer values as they move further away from the IGF sequence. For example, amino acid positions within a dipeptide prodrug linked to the N-terminus of IGF are designated aa-1 -aa0 -IGF, where aa0 represents the carboxy-terminal amino acid of the dipeptide and aa-1 represents the amino-terminal amino acid of the dipeptide.
本文使用的术语“羟酸”是指已经修饰以用羟基置换α碳氨基的氨基酸。As used herein, the term "hydroxy acid" refers to an amino acid that has been modified to replace the alpha-carbon amino group with a hydroxyl group.
本文使用的术语“非编码氨基酸”包括不是任何下列20个氨基酸的L-异构体的任何氨基酸：Ala、Cys、Asp、Glu、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Thr、Val、Trp、Tyr。As used herein, the term "non-coded amino acid" includes any amino acid that is not the L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr.
“二肽”是α氨基酸或α羟酸通过肽键与另一个氨基酸连接形成的化合物。A "dipeptide" is a compound formed by linking an alpha amino acid or alpha hydroxy acid to another amino acid via a peptide bond.
本文使用的不存在任何其它指称的术语“化学裂解”包括导致共价化学键断裂的非酶促反应。The term "chemical cleavage" as used herein without any other reference includes non-enzymatic reactions that result in the breaking of covalent chemical bonds.
“生物活性多肽”是指能够体外和/或体内施加生物作用的多肽。"Biologically active polypeptide" refers to a polypeptide capable of exerting a biological effect in vitro and/or in vivo.
本文所用对肽的一般提及意在包括具有修饰的氨基端和羧基端的肽。例如，标明标准氨基酸的氨基酸序列意在包括N端和C端上的标准氨基酸以及N端上相应的羟酸和/或经修饰包含替换末端羧酸的酰胺基的相应的C端氨基酸。Generic references to peptides as used herein are intended to include peptides with modified amino and carboxy termini. For example, an amino acid sequence indicating a standard amino acid is intended to include the standard amino acid at the N-terminus and the C-terminus as well as the corresponding hydroxyacid at the N-terminus and/or the corresponding C-terminal amino acid modified to contain an amide group in place of the terminal carboxylic acid.
本文使用的“酰化的”氨基酸是包含对天然存在的氨基酸而言是非天然的酰基的氨基酸，与其制备方法无关。产生酰化氨基酸和酰化肽的示例性方法是本领域已知的，该方法包括在包含入肽前使氨基酸酰化或在肽合成之后接着对肽进行化学酰化。在一个实施方案中，酰基导致肽具有一种或多种以下性质：(i)循环中的半寿期延长、(ii)起效延迟、(iii)作用持续时间延长、(iv)对蛋白酶(例如DPP-IV)的抗性改进、和(v)对胰岛素肽受体的效能增加。As used herein, an "acylated" amino acid is one that contains an acyl group that is unnatural to a naturally occurring amino acid, regardless of its method of preparation. Exemplary methods of producing acylated amino acids and acylated peptides are known in the art and include acylation of the amino acid prior to incorporation into the peptide or subsequent chemical acylation of the peptide following peptide synthesis. In one embodiment, the acyl group results in a peptide with one or more of the following properties: (i) increased half-life in circulation, (ii) delayed onset of action, (iii) increased duration of action, (iv) sensitivity to proteases ( For example DPP-IV) improved resistance, and (v) increased potency at the insulin peptide receptor.
本文使用的“烷基化的”氨基酸是包含对天然存在的氨基酸而言是非天然的烷基的氨基酸，与其制备方法无关。产生烷基化氨基酸和烷基化肽的示例性方法是本领域已知的，该方法包括在包含入肽中之前使氨基酸烷基化或肽合成之后接着对肽进行化学烷基化。在不固守任何特定理论的情况下，据认为肽的烷基化将获得与肽的酰化相似(如果不是相同的话)的作用，例如循环中的半寿期延长、起效延迟、作用持续时间延长、对蛋白酶(例如DPP-IV)的抗性改进和对胰岛素肽受体效能增加。As used herein, an "alkylated" amino acid is an amino acid that contains an alkyl group that is unnatural to a naturally occurring amino acid, regardless of its method of preparation. Exemplary methods of producing alkylated amino acids and alkylated peptides are known in the art and include alkylation of amino acids prior to incorporation into peptides or subsequent chemical alkylation of peptides following peptide synthesis. Without being bound to any particular theory, it is believed that alkylation of peptides will achieve similar, if not identical, effects as acylation of peptides, such as increased half-life in circulation, delayed onset of action, duration of action Prolongation, improved resistance to proteases (eg DPP-IV) and increased potency at the insulin peptide receptor.
本文使用的术语“药学上可接受的载体”包括任何标准的药用载体，例如磷酸缓冲盐溶液、水、乳剂(例如油/水或水/油乳剂)和各种类型的润湿剂。该术语还包括美国联邦政府管理机构批准或美国药典录入的用于动物(包括人)的任何物质。As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions (eg, oil/water or water/oil emulsions) and various types of wetting agents. The term also includes any substance approved by a regulatory agency of the United States Federal Government or listed in the United States Pharmacopoeia for use in animals, including humans.
本文使用的术语“药学上可接受的盐”是指保留母体化合物的生物活性的化合物的盐，并且其在生物学或其它方面不是不合乎需要的。本文公开的许多化合物能够通过氨基和/或羧基或其类似基团的存在而形成酸式盐和/或碱式盐。As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that retains the biological activity of the parent compound and which is not biologically or otherwise undesirable. Many of the compounds disclosed herein are capable of forming acid and/or base salts through the presence of amino and/or carboxyl groups or the like.
可由无机和有机碱制备药学上可接受的碱加成盐。仅作为实例，由无机碱得到的盐包括钠盐、钾盐、锂盐、铵盐、钙盐和镁盐。由有机碱得到的盐包括但不限于伯胺、仲胺和叔胺的盐。Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. By way of example only, salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines.
可由无机和有机酸制备药学上可接受的酸加成盐。由无机酸得到的盐包括盐酸、氢溴酸、硫酸、硝酸、磷酸等的盐。由有机酸得到的盐包括乙酸、丙酸、乙醇酸、丙酮酸、草酸、苹果酸、丙二酸、琥珀酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸等的盐。Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Salts derived from inorganic acids include salts of hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Salts derived from organic acids include acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, almond acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.
本文使用的术语“治疗”包括预防特定的病症或病况，或减轻特定病症或病况的相关症状和/或防止或消除所述症状。例如，本文使用的术语“治疗糖尿病”总的来说是指保持葡萄糖血液水平接近正常水平，并可根据既定情况，包括提高或降低血糖水平。As used herein, the term "treating" includes preventing the particular disorder or condition, or alleviating the symptoms associated with a particular disorder or condition and/or preventing or eliminating the symptoms. For example, the term "treating diabetes" as used herein generally refers to maintaining glucose blood levels near normal levels and may include raising or lowering blood sugar levels, depending on the given situation.
本文使用的“有效的”量或“治疗有效量”的前药是指无毒但足量的前药以提供所需作用。例如一种所需要的作用会是预防或治疗高血糖症。“有效”量在受试者之间不同，这取决于个体的年龄和一般状况、给药方式等。因此，并非总是能指定确切的“有效量”。然而，在任何个体情况下，本领域普通技术人员采用常规实验可确定合适的“有效”量。As used herein, an "effective" amount or "therapeutically effective amount" of a prodrug refers to a non-toxic but sufficient amount of the prodrug to provide the desired effect. For example one desired effect would be the prevention or treatment of hyperglycemia. An "effective" amount will vary from subject to subject, depending on the age and general condition of the individual, the mode of administration, and the like. Thus, an exact "effective amount" cannot always be specified. However, an appropriate "effective" amount can be determined in any individual case by one of ordinary skill in the art using routine experimentation.
术语“胃肠外”意指不通过消化道但通过某些其它途径，例如鼻内、吸入、皮下、肌内、脊柱内或静脉内。The term "parenteral" means not via the alimentary canal but via some other route, eg intranasally, inhalationally, subcutaneously, intramuscularly, intraspinally or intravenously.
本文使用的术语“天然胰岛素肽”意在表示包含A链SEQ ID NO：1和B链SEQ ID NO：2的51个氨基酸异二聚体以及包含SEQ ID NO：1和2的单链胰岛素类似物。本文使用的缺乏更多描述性语言的术语“胰岛素肽”意在包括包含A链SEQ ID NO：1和B链SEQ ID NO：2的51个氨基酸异二聚体及其单链胰岛素类似物(包括例如已公布的国际申请WO96/34882和美国专利号6,630,348中公开的单链胰岛素类似物，其公开内容通过引用结合到本文中)，以及包括包含天然A链和/或B链的修饰衍生物的异二聚体和单链类似物，所述修饰衍生物包括A19、B16或B25位上的氨基酸被修饰成4-氨基苯丙氨酸，或者一个或多个选自以下位置的氨基酸取代：A5、A8、A9、A10、A12、A14、A15、A17、A18、A21、B1、B2、B3、B4、B5、B9、B 10、B13、B14、B17、B20、B21、B22、B23、B26、B27、B28、B29和B30，或者B1-4和B26-30位置中的任何或全部的缺失。本文使用的“胰岛素前药类似物”是指在干扰胰岛素的活性或基于IGF1的胰岛素类似物的活性(例如与胰岛素和IGF-1受体相互作用的能力)的位置上，通过经由酰胺键共价连接二肽来修饰的胰岛素肽(或实施例9中公开的基于IGF1的胰岛素类似物)。The term "native insulin peptide" as used herein is intended to mean a 51 amino acid heterodimer comprising the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2 and a single chain insulin analogue comprising SEQ ID NO: 1 and 2 things. The term "insulin peptide" used herein in the absence of more descriptive language is intended to include a 51 amino acid heterodimer comprising the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2 and its single chain insulin analogue ( Including, for example, single-chain insulin analogs disclosed in Published International Application WO96/34882 and U.S. Patent No. 6,630,348, the disclosures of which are incorporated herein by reference), as well as modified derivatives comprising native A chain and/or B chain The heterodimer and single-chain analogs of the modified derivatives include that the amino acid at position A19, B16 or B25 is modified to 4-aminophenylalanine, or one or more amino acid substitutions selected from the following positions: A5, A8, A9, A10, A12, A14, A15, A17, A18, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B17, B20, B21, B22, B23, B26 , B27, B28, B29 and B30, or any or all of the B1-4 and B26-30 positions. As used herein, "insulin prodrug analogue" refers to, at a position that interferes with the activity of insulin or the activity of an IGF1-based insulin analogue (such as the ability to interact with insulin and the IGF-1 receptor) Insulin peptides (or IGF1-based insulin analogs disclosed in Example 9) modified by linking dipeptides.
本文使用的术语“单链胰岛素类似物”包括一组结构上相关的蛋白质，其中胰岛素A和B链共价连接。The term "single-chain insulin analog" as used herein includes a group of structurally related proteins in which the A and B chains of insulin are covalently linked.
本文使用的氨基酸“修饰”是指氨基酸的取代、添加或缺失，或者通过在/从氨基酸上添加和/或除去化学基团而对氨基酸的衍生，包括用任何通常存在于人蛋白质中的20个氨基酸以及非典型的或非天然存在的氨基酸取代或添加任何所述氨基酸。非典型氨基酸的商用来源包括Sigma-Aldrich(Milwaukee，WI)，ChemPep Inc.(Miami，FL)和GenzymePharmaceuticals(Cambridge，MA)。非典型氨基酸可购自商业供应商、从头合成、或者由天然存在的氨基酸进行化学修饰或衍生化。Amino acid "modification" as used herein refers to the substitution, addition or deletion of amino acids, or the derivatization of amino acids by adding and/or removing chemical groups to/from amino acids, including any of the 20 Amino acids as well as atypical or non-naturally occurring amino acids are substituted for or added to any such amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA). Atypical amino acids can be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from naturally occurring amino acids.
本文使用的氨基酸“取代”是指一个氨基酸残基被不同的氨基酸残基置换。在本申请全文中，所有通过字母和数字提及的具体氨基酸位置(例如A5位)是指在各自天然人胰岛素A链(SEQ ID NO：1)或B链(SEQ ID NO：2)中A链(例如A5位)或B链(例如B5位)的该位置上的氨基酸，或其任何类似物中相应的氨基酸位置。例如，本文缺乏任何更多详尽描述提及的“B28位”意指其中缺失了SEQ ID NO：2的第一个氨基酸的胰岛素类似物B链相应的B27位。Amino acid "substitution" as used herein refers to the replacement of one amino acid residue with a different amino acid residue. Throughout this application, all references to specific amino acid positions by letters and numbers (eg position A5) refer to A in the respective native human insulin A chain (SEQ ID NO: 1) or B chain (SEQ ID NO: 2). The amino acid at that position in the chain (eg, position A5) or the B chain (eg, position B5), or the corresponding amino acid position in any analog thereof. For example, reference herein to "position B28" in the absence of any further elaboration means the corresponding position B27 of the insulin analog B chain in which the first amino acid of SEQ ID NO: 2 is deleted.
将本文使用的术语“保守氨基酸取代”在此定义为在下列5组之一内的互换：The term "conservative amino acid substitution" as used herein is defined herein as an exchange within one of the following 5 groups:
I.小的脂族非极性或略有极性的残基：I. Small aliphatic nonpolar or slightly polar residues:
Ala、Ser、Thr、Pro、Gly；Ala, Ser, Thr, Pro, Gly;
II.极性带负电荷的残基及其酰胺：II. Polar negatively charged residues and their amides:
Asp、Asn、Glu、Gln；Asp, Asn, Glu, Gln;
III.极性带正电荷的残基：III. Polar positively charged residues:
His、Arg、Lys、鸟氨酸(Orn)；His, Arg, Lys, ornithine (Orn);
IV.大的脂族非极性残基：IV. Large aliphatic nonpolar residues:
Met、Leu、Ile、Val、Cys、正亮氨酸(Nle)、高半胱氨酸；Met, Leu, Ile, Val, Cys, Norleucine (Nle), Homocysteine;
V.大的芳族残基：V. Large Aromatic Residues:
Phe、Tyr、Trp、乙酰基苯丙氨酸。Phe, Tyr, Trp, Acetyl Phenylalanine.
本文使用的通用术语“聚乙二醇链”或“PEG链”是指呈支链或直链的环氧乙烷和水的缩聚物的混合物，用通式H(OCH2CH2)nOH表示，其中n至少为9。缺乏任何更多描述的该术语意在包括平均总分子量为500-80,000道尔顿的乙二醇聚合物。“聚乙二醇链”或“PEG链”与数字后缀联用以表示其大致平均分子量。例如，PEG-5,000是指总分子量平均为约5,000道尔顿的聚乙二醇链。The general term "polyethylene glycol chain" or "PEG chain" as used herein refers to a mixture of branched or linear polycondensates of ethylene oxide and water, represented by the general formula H(OCH2 CH2 )n OH where n is at least 9. The term, lacking any further description, is intended to include ethylene glycol polymers having an average overall molecular weight of 500-80,000 Daltons. "Polyethylene glycol chain" or "PEG chain" is used with a numerical suffix to indicate its approximate average molecular weight. For example, PEG-5,000 refers to polyethylene glycol chains having an average total molecular weight of about 5,000 Daltons.
本文使用的术语“聚乙二醇化的”等术语是指已通过将聚乙二醇链与化合物连接对其天然状态进行修饰的化合物。“聚乙二醇化多肽”是具有与多肽共价结合的PEG链的多肽。As used herein, the term "pegylated" and like terms refer to a compound that has been modified from its native state by attaching polyethylene glycol chains to the compound. A "PEGylated polypeptide" is a polypeptide that has a PEG chain covalently bound to the polypeptide.
本文使用的“接头”是使两个独立的实体彼此结合的键、分子或分子的基团。接头可为两个实体提供最佳间隔，或者还可提供使两个实体彼此分开的不稳定连接。不稳定连接包括光可裂解基团、酸不稳定部分、碱不稳定部分和酶可切割基团。As used herein, a "linker" is a bond, molecule or group of molecules that joins two separate entities to each other. Joints may provide optimal separation of two entities, or may also provide an unstable connection that separates two entities from each other. Labile linkages include photocleavable groups, acid labile moieties, base labile moieties, and enzyme cleavable groups.
本文使用的“胰岛素二聚体”是包含2个胰岛素肽通过接头彼此共价结合的复合体。在缺乏任何限定性语言下使用时，术语胰岛素二聚体既包括胰岛素同二聚体又包括胰岛素异二聚体。胰岛素同二聚体包含2个相同的亚基(各自包含A链和B链)，而胰岛素异二聚体包含2个不同的亚基，然而2个亚基彼此大致相似。An "insulin dimer" as used herein is a complex comprising 2 insulin peptides covalently bound to each other via a linker. When used in the absence of any limiting language, the term insulin dimer includes both insulin homodimers and insulin heterodimers. Insulin homodimers comprise 2 identical subunits (each comprising an A chain and a B chain), while insulin heterodimers comprise 2 different subunits, however the 2 subunits are roughly similar to each other.
本文使用的术语“C1-Cn烷基”(其中n可为1-6)表示具有一个至指定数目的碳原子的支链或直链烷基。通常C1-C6烷基包括但不限于甲基、乙基、正丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、戊基、己基等。The term "C1 -Cn alkyl" (wherein n can be 1-6) as used herein denotes a branched or straight chain alkyl group having from one to the specified number of carbon atoms. Typical C1 -C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like.
本文使用的术语“C2-Cn烯基”(其中n可为2-6)表示具有2个至指定数目的碳原子和至少一个双键的烯属不饱和支链或直链基团。这类基团的实例包括但不限于1-丙烯基、2-丙烯基(-CH2-CH＝CH2)、1，3-丁二烯基、(-CH＝CHCH＝CH2)、1-丁烯基(-CH＝CHCH2CH3)、己烯基、戊烯基等。The term "C2 -Cn alkenyl" (wherein n can be 2-6) as used herein denotes an ethylenically unsaturated branched or straight chain group having 2 to the indicated number of carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, 1-propenyl, 2-propenyl (-CH2 -CH=CH2 ), 1,3-butadienyl, (-CH=CHCH=CH2 ), 1 -butenyl (-CH=CHCH2 CH3 ), hexenyl, pentenyl and the like.
术语“C2-Cn炔基”(其中n可为2-6)是指具有2-n个碳原子和至少一个三键的不饱和支链或直链基团。这类基团的实例包括但不限于1-丙炔基、2-丙炔基、1-丁炔基、2-丁炔基、1-戊炔基等。The term "C2 -Cn alkynyl" (where n can be 2-6) refers to an unsaturated branched or straight chain group having 2-n carbon atoms and at least one triple bond. Examples of such groups include, but are not limited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the like.
本文使用的术语“芳基”是指具有一个或两个芳环的单环或二环碳环系统，包括但不限于苯基、萘基、四氢萘基、茚满基、茚基等。通过指明碳存在的数目来表示芳环的大小和取代基或连接基团的存在。例如，术语“(C1-C3烷基)(C6-C10芳基)”是指通过1-3元烷基链与母体部分连接的5-10元芳基。The term "aryl" as used herein refers to a monocyclic or bicyclic carbocyclic ring system having one or two aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. The size of the aromatic ring and the presence of substituents or linking groups are indicated by indicating the number of carbons present. For example, the term "(C1 -C3 alkyl)(C6 -C10 aryl)" refers to a 5-10 membered aryl group attached to the parent moiety through a 1-3 membered alkyl chain.
本文使用的术语“杂芳基”是指含有一个或两个芳环并在芳环中含有至少一个氮、氧或硫原子的单环或二环环系。通过指明碳存在的数目来表示杂芳环的大小和取代基或连接基团的存在。例如，术语“(C1-Cn烷基)(C5-C6杂芳基)”是指通过1-“n”元烷基链与母体部分连接的5或6元杂芳基。As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and at least one nitrogen, oxygen or sulfur atom in the aromatic rings. The size of the heteroaryl ring and the presence of substituents or linking groups are indicated by indicating the number of carbons present. For example, the term "(C1 -Cnalkyl )(C5 -C6heteroaryl)" refers to a 5- or6 -membered heteroaryl group attached to the parent moiety through a 1-"n" membered alkyl chain.
术语“C3-Cn环烷基”是指包含碳和氢原子的非芳族单环或多环，其下标数字表示碳原子存在的数目。例如术语C3-C8环烷基表示环丙基、环丁基、环戊基、环己基、环庚基和环辛基化合物。The term "C3 -Cn cycloalkyl" refers to a non-aromatic monocyclic or polycyclic ring comprising carbon and hydrogen atoms, the subscript number indicating the number of carbon atoms present. For example the term C3 -C8 cycloalkyl denotes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl compounds.
术语“C3-Cn杂环基”是指含有1-“n-1”个杂原子的环烷基环系，其中杂原子选自氧、硫和氮。例如措词“5元杂环”或“C5杂环”包括但不限于具有一个杂原子的5元杂环(例如噻吩、吡咯、呋喃)；在1，2或1，3位具有两个杂原子的5元杂环(例如唑、吡唑、咪唑、噻唑、嘌呤)；具有3个杂原子的5元杂环(例如三唑、噻二唑)。The term "C3-Cnheterocyclyl " refers to a cycloalkyl ring system containing 1 to "n-1" heteroatoms selected from oxygen, sulfur and nitrogen. For example, the phrase "5-membered heterocycle" or "C5 heterocycle" includes, but is not limited to, 5-membered heterocycles with one heteroatom (e.g., thiophene, pyrrole, furan); 5-membered heterocycles with heteroatoms (e.g. azoles, pyrazoles, imidazoles, thiazoles, purines); 5-membered heterocycles with 3 heteroatoms (eg triazoles, thiadiazoles).
本文使用的术语“C3-Cn元环”是指包含共3-“n”个数目的元素彼此连接形成环的饱和或不饱和烃环结构，其中环元素选自C、O、S和N。该术语意在包括环烷基、杂环、芳基和杂芳基。The term "C3 -Cn -membered ring" as used herein refers to a saturated or unsaturated hydrocarbon ring structure comprising a total of 3-"n" number of elements linked to each other to form a ring, wherein the ring elements are selected from C, O, S and N. The term is intended to include cycloalkyl, heterocycle, aryl and heteroaryl.
本文使用的术语“卤素”是指选自氟、氯、溴和碘的一个或多个成员。As used herein, the term "halogen" refers to one or more members selected from fluorine, chlorine, bromine and iodine.
本文使用的无更多规定的术语“患者”意在包括任何驯养的温血脊椎动物(包括例如但不限于家畜、马、猫、狗和其它宠物)和人。The term "patient" as used herein without further specification is intended to include any domesticated warm-blooded vertebrate (including, for example, but not limited to, livestock, horses, cats, dogs, and other pets) and humans.
实施方案implementation plan
本公开内容提供配制成延迟起效和延长胰岛素肽的半寿期，从而改进基础胰岛素肽的治疗指数的胰岛素前药衍生物。本文公开的胰岛素前药化学法允许通过非酶促降解机制激活前药。所公开的前药化学法可与活性部位胺化学缀合形成酰胺，酰胺在二酮哌嗪形成时恢复成为母体胺并释放前药元件。这种新的生物友好型前药化学法在生理条件(例如pH约7，37℃，水性环境)下自发降解，而且不依赖于酶促降解。前药衍生物的持续时间取决于二肽前药序列的选择，因此为前药制剂提供了灵活性。The present disclosure provides insulin prodrug derivatives formulated to delay onset of action and prolong the half-life of insulin peptides, thereby improving the therapeutic index of basal insulin peptides. The insulin prodrug chemistry disclosed herein allows activation of the prodrug through non-enzymatic degradation mechanisms. The disclosed prodrug chemistry allows for chemical conjugation of the active site amine to form an amide which, upon formation of the diketopiperazine, reverts to the parent amine and releases the prodrug element. This new biofriendly prodrug chemistry degrades spontaneously under physiological conditions (eg, pH around 7, 37°C, aqueous environment) and does not rely on enzymatic degradation. The duration of prodrug derivatives depends on the choice of dipeptide prodrug sequence, thus providing flexibility in prodrug formulation.
在一个实施方案中，提供在生理条件下非酶促激活半寿期(t1/2)介于1-100小时之间的前药。本文公开的生理条件意在包括温度为约35-40℃，pH为约7.0-约7.4，更通常包括水性环境中pH为7.2-7.4，温度为36-38℃。在一个实施方案中，在生理条件下能够形成二酮哌嗪的二肽通过酰胺键与胰岛素肽共价连接。In one embodiment, there is provided a prodrug with a non-enzymatic activation half-life (t1/2) between 1-100 hours under physiological conditions. Physiological conditions disclosed herein are meant to include a temperature of about 35-40°C and a pH of about 7.0 to about 7.4, and more typically include a pH of 7.2-7.4 and a temperature of 36-38°C in an aqueous environment. In one embodiment, a dipeptide capable of forming a diketopiperazine under physiological conditions is covalently linked to the insulin peptide via an amide bond.
有利的是，裂解速率(由此激活前药)取决于二肽前部分(pro-moiety)的结构和立体化学，还取决于亲核体的强度。本文公开的前药最终将以化学法转化成可被该药物的天然受体识别的结构，其中该化学转化的速度将决定体内生物作用的开始时间和持续时间。本申请公开的前药化学法有赖于不依赖额外化学添加剂或酶的分子内化学反应。转化速度受二肽取代基的化学性质和在生理条件下二肽取代基的裂解所控制。由于生理pH和温度紧紧地调节在严格限定的范围内，因此前药至药物的转化速度表现出高的患者内和患者间再现性。Advantageously, the rate of cleavage (and thus activation of the prodrug) depends on the structure and stereochemistry of the dipeptide pro-moiety, and also on the strength of the nucleophile. The prodrugs disclosed herein will ultimately be chemically converted to structures recognized by the drug's natural receptors, wherein the rate of this chemical conversion will determine the onset and duration of biological effects in vivo. The prodrug chemistry disclosed in this application relies on intramolecular chemical reactions that do not rely on additional chemical additives or enzymes. The rate of conversion is controlled by the chemical nature of the dipeptide substituent and the cleavage of the dipeptide substituent under physiological conditions. Since physiological pH and temperature are tightly regulated within tightly defined ranges, the rate of prodrug-to-drug conversion exhibits high intra- and inter-patient reproducibility.
如本文所公开，提供前药，其中生物活性多肽具有至少1小时、更通常大于20小时但小于100小时的延长半寿期，并且在生理条件下，通过由固有的化学不稳定性驱动的非酶促反应而转变为活性形式。在一个实施方案中，前药的非酶促活化t1/2时间介于1-100小时之间，更通常介于12小时和72小时之间，而在一个实施方案中，通过将前药在磷酸盐缓冲溶液(例如PBS)中于37℃和pH 7.2下保温测定的t1/2介于24-48小时之间。运用公式t1/2＝.693/k，其中‘k’为前药降解的一级速率常数，来计算各种前药的半寿期。在一个实施方案中，前药的活化在酰胺键连接的二肽裂解，并形成二酮哌嗪或二酮吗啉和活性胰岛素肽之后发生。As disclosed herein, prodrugs are provided in which the biologically active polypeptide has an extended half-life of at least 1 hour, more typically greater than 20 hours but less than 100 hours, and under physiological conditions, is activated by non-toxicity driven by inherent chemical instability. Converted to the active form by an enzymatic reaction. In one embodiment, the non-enzymatic activation t1/2 time of the prodrug is between 1-100 hours, more typically between 12 hours and 72 hours, and in one embodiment, the prodrug is The t1/2 measured by incubation in phosphate buffered saline (eg PBS) at 37°C and pH 7.2 ranges between 24-48 hours. The half-lives of the various prodrugs were calculated using the formula t1/2 =.693/k, where 'k' is the first order rate constant for prodrug degradation. In one embodiment, activation of the prodrug occurs following cleavage of the amide-linked dipeptide and formation of the diketopiperazine or diketomorpholine and the active insulin peptide.
已经证实由天然或合成氨基酸组成的特定二肽在生理条件下促进分子内分解以释放活性胰岛素肽。二肽可与存在于天然胰岛素的氨基或者与通过天然胰岛素肽修饰而引入胰岛素肽的氨基连接(通过酰胺键)。在一个实施方案中，选择抗存在于哺乳动物血清的肽酶(包括例如二肽基肽酶IV(DPP-IV))切割的二肽结构。因此，在一个实施方案中，与蛋白酶不存在时进行的反应相比，在血清蛋白酶存在下利用生理条件进行反应时，二肽前药元件从生物活性肽上的裂解速率不显著提高(例如大于2X)。因此二肽前药元件自胰岛素肽的裂解半寿期(在生理条件下，在PBS中)不超过在包含DPP-IV蛋白酶的溶液中二肽前药元件自胰岛素肽的裂解半寿期的2、3、4或5倍。在一个实施方案中，包含DPP-IV蛋白酶的溶液为血清，更具体地讲为哺乳动物血清，包括人血清。Specific dipeptides composed of natural or synthetic amino acids have been shown to promote intramolecular breakdown to release active insulin peptides under physiological conditions. The dipeptide can be linked (via an amide bond) to an amino group present in native insulin or to an amino group introduced into the insulin peptide by modification of the native insulin peptide. In one embodiment, dipeptide structures are selected that resist cleavage by peptidases present in mammalian serum, including, for example, dipeptidyl peptidase IV (DPP-IV). Thus, in one embodiment, the rate of cleavage of a dipeptide prodrug element from a biologically active peptide is not significantly increased (e.g., greater than 2X). Thus the cleavage half-life of the dipeptide prodrug element from the insulin peptide (under physiological conditions, in PBS) does not exceed 2 times the cleavage half-life of the dipeptide prodrug element from the insulin peptide in a solution containing DPP-IV protease. , 3, 4 or 5 times. In one embodiment, the solution comprising DPP-IV protease is serum, more specifically mammalian serum, including human serum.
根据一个实施方案，二肽前药元件包含结构U-O，其中U为氨基酸或羟酸，O为N-烷基化氨基酸。在一个实施方案中，U、O或U-O与之连接的胰岛素肽的氨基酸为非编码氨基酸。在一个实施方案中，U和/或O为呈D立体异构体构型的氨基酸。在一些示例性的实施方案中，U为呈D立体异构体构型的氨基酸，而O为呈L立体异构体构型的氨基酸。在一些示例性的实施方案中，U为呈L立体异构体构型的氨基酸，而O为呈D立体异构体构型的氨基酸。在一些示例性的实施方案中，U为呈D立体异构体构型的氨基酸，而O为呈D立体异构体构型的氨基酸。在一个实施方案中，O为N-烷基化氨基酸但不是脯氨酸。在一个实施方案中，氨基酸O的N-烷基化基团为C1-C18烷基，在一个实施方案中，N-烷基化基团为C1-C6烷基。在一个实施方案中，U-O为包含本文定义的式I结构的二肽。According to one embodiment, the dipeptide prodrug element comprises the structure UO, wherein U is an amino acid or hydroxy acid and O is an N-alkylated amino acid. In one embodiment, the amino acid of the insulin peptide to which the U, O, or UO is linked is a non-coded amino acid. In one embodiment, U and/or O are amino acids in the D stereoisomer configuration. In some exemplary embodiments, U is an amino acid in the D stereoisomer configuration and O is an amino acid in the L stereoisomer configuration. In some exemplary embodiments, U is an amino acid in the L stereoisomer configuration and O is an amino acid in the D stereoisomer configuration. In some exemplary embodiments, U is an amino acid in the D stereoisomer configuration and O is an amino acid in the D stereoisomer configuration. In one embodiment, O is an N-alkylated amino acid but not proline. In one embodiment, the N-alkylated group of amino acid O is C1 -C18 alkyl, and in one embodiment, the N-alkylated group is C1 -C6 alkyl. In one embodiment, UO is a dipeptide comprising the structure of formula I as defined herein.
在一个实施方案中，二肽在选自A链或B链的N端氨基的氨基上或者在存在于胰岛素肽活性部位的氨基酸的侧链氨基上与胰岛素肽连接。根据一个实施方案，二肽延伸物通过存在于或接近活性部位的赖氨酸残基的侧链胺与胰岛素肽共价连接。在一个实施方案中，二肽延伸物通过合成氨基酸或修饰氨基酸连接，其中合成氨基酸或修饰氨基酸具有适于二肽延伸物共价连接的官能团(例如氨基-苯丙氨酸的芳族胺)。根据一个实施方案，二肽在选自A链或B链的N端氨基的氨基上或者在存在于A19、B16或B25位的芳族胺(例如4-氨基-苯丙氨酸残基)的侧链氨基上与胰岛素肽连接。在一个实施方案中，U-O二肽在A19位通过存在于A19位的4-氨基苯丙氨酸结合。In one embodiment, the dipeptide is linked to the insulin peptide at an amino group selected from the N-terminal amino group of the A chain or B chain or at the side chain amino group of an amino acid present in the active site of the insulin peptide. According to one embodiment, the dipeptide extension is covalently linked to the insulin peptide via the side chain amine of a lysine residue present at or near the active site. In one embodiment, the dipeptide extension is attached via a synthetic or modified amino acid having a functional group suitable for covalent attachment of the dipeptide extension (eg, an aromatic amine of amino-phenylalanine). According to one embodiment, the dipeptide is on an amino group selected from the N-terminal amino group of chain A or B or on an aromatic amine present at position A19, B16 or B25 (eg 4-amino-phenylalanine residue) The amino group of the side chain is connected to the insulin peptide. In one embodiment, the U-O dipeptide is bound at position A19 through the 4-aminophenylalanine present at position A19.
将二肽前药元件设计成在生理条件下和在缺乏酶活性时自发裂解其酰胺键成为胰岛素类似物。在一个实施方案中，二肽延伸物的N端氨基酸包含C-烷基化氨基酸(例如氨基异丁酸)。在一个实施方案中，二肽的C端氨基酸包含N-烷基化氨基酸(例如脯氨酸或N-甲基甘氨酸)。在一个实施方案中，二肽包含N端C-烷基化氨基酸和随后的N-烷基化氨基酸的序列。The dipeptide prodrug element is designed to spontaneously cleave its amide bond to an insulin analog under physiological conditions and in the absence of enzymatic activity. In one embodiment, the N-terminal amino acid of the dipeptide extension comprises a C-alkylated amino acid (eg, aminoisobutyric acid). In one embodiment, the C-terminal amino acid of the dipeptide comprises an N-alkylated amino acid (eg, proline or N-methylglycine). In one embodiment, the dipeptide comprises a sequence of N-terminal C-alkylated amino acids followed by N-alkylated amino acids.
申请人发现，在A链19位上的天然酪氨酸上可以容纳选择性插入的4-氨基苯基氨基酸部分而又不损失胰岛素肽的效能(参见图3)。该活性部位氨基随后与本文公开的二肽前药部分进行的化学酰胺化显著降低胰岛素受体结合活性，从而提供胰岛素的合适前药(参见图6，给出IGF1Y16L17(p-NH2-F)A19类似物的数据，已证实该类似物具有与胰岛素(p-NH2-F)A19相当的活性，参见图4)。因此，在一个实施方案中，二肽前药元件通过酰胺键与A194-氨基苯丙氨酸的芳环连接，其中二肽的C端氨基酸包含N-烷基化氨基酸，而二肽的N端氨基酸为任何氨基酸。Applicants have found that an optional insertion of a 4-aminophenyl amino acid moiety can be accommodated at the native tyrosine at position 19 of the A chain without loss of insulin peptide potency (see Figure 3). Subsequent chemical amidation of this active site amino group with the dipeptide prodrug moiety disclosed herein significantly reduces insulin receptor binding activity, thereby providing a suitable prodrug of insulin (see Figure 6, given IGF1Y16 L17 (p-NH2- F) Data for the analogue of A19, which has been shown to have comparable activity to insulin (p-NH2 -F)A19 , see Figure 4). Thus, in one embodiment, the dipeptide prodrug element is linked via an amide bond to the aromatic ring of A194-aminophenylalanine, wherein the C-terminal amino acid of the dipeptide contains an N-alkylated amino acid and the N-terminal of the dipeptide An amino acid is any amino acid.
二肽前药部分还可与胰岛素肽的其它部位连接以制备胰岛素的前药或长效类似物(depot analog)。根据一个实施方案，提供胰岛素前药/长效类似物，其包含A链、B链和通过酰胺键与选自以下的一个或多个位点连接的二肽：A链或B链的N端氨基或者内部氨基酸的侧链氨基，包括例如与存在于A19、B16或B25位上的4-氨基-苯丙氨酸残基的芳族胺连接的二肽。在一个实施方案中，胰岛素肽包含两个二肽元件，其中二所述肽元件任选聚乙二醇化、烷基化、酰化或与长效聚合物连接。在一个实施方案中，二肽包含后接N-烷基化氨基酸的N端C-烷基化氨基酸。The dipeptide prodrug moiety can also be linked to other parts of the insulin peptide to make a prodrug or depot analog of insulin. According to one embodiment, there is provided an insulin prodrug/long-acting analogue comprising an A chain, a B chain and a dipeptide linked by an amide bond to one or more sites selected from: the N-terminus of the A chain or the B chain Amino groups or side chain amino groups of internal amino acids, include for example dipeptides linked to the aromatic amine of a 4-amino-phenylalanine residue present at position A19, B16 or B25. In one embodiment, the insulin peptide comprises two dipeptide elements, wherein both said peptide elements are optionally pegylated, alkylated, acylated or linked to a long-acting polymer. In one embodiment, the dipeptide comprises an N-terminal C-alkylated amino acid followed by an N-alkylated amino acid.
包含胰岛素前药类似物的A链和B链可包含各自肽的天然序列(即SEQ ID NO：1和SEQ ID NO：2)，或者可包含SEQ ID NO：1和/或SEQ ID NO：2的衍生物，其中所述衍生物包括将A19、B16或B25位上的氨基酸修饰成4-氨基苯丙氨酸和/或一个或多个选自以下位置的氨基酸取代：A5、A8、A9、A10、A14、A15、A17、A18、A19和A21、B1、B2、B3、B4、B5、B9、B10、B13、B14、B 17、B20、B22、B23、B26、B27、B28、B29和B30；或者在B1-4和B26-30位置中的任何或全部上的缺失。在一个实施方案中，二肽前药元件与A链或B链的N端氨基连接，其中二肽前药元件的C端氨基酸包含N-烷基化氨基酸，而二肽前药元件的N端氨基酸为任何氨基酸，前提条件是如果二肽的C端氨基酸为脯氨酸，则二肽的N端氨基酸包含C-烷基化氨基酸。The A and B chains comprising the insulin prodrug analog may comprise the native sequences of the respective peptides (i.e. SEQ ID NO: 1 and SEQ ID NO: 2), or may comprise SEQ ID NO: 1 and/or SEQ ID NO: 2 derivatives, wherein the derivatives include modifying the amino acid at A19, B16 or B25 to 4-aminophenylalanine and/or one or more amino acid substitutions selected from the following positions: A5, A8, A9, A10, A14, A15, A17, A18, A19 and A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B17, B20, B22, B23, B26, B27, B28, B29 and B30 ; or a deletion at any or all of the B1-4 and B26-30 positions. In one embodiment, the dipeptide prodrug element is linked to the N-terminal amino group of chain A or B, wherein the C-terminal amino acid of the dipeptide prodrug element comprises an N-alkylated amino acid, and the N-terminal amino acid of the dipeptide prodrug element The amino acid is any amino acid, provided that if the C-terminal amino acid of the dipeptide is proline, the N-terminal amino acid of the dipeptide comprises a C-alkylated amino acid.
在一个实施方案中，二肽前药元件包含下式I的通用结构：In one embodiment, the dipeptide prodrug element comprises the general structure of Formula I below:
其中in
R1、R2、R4和R8独立选自H、C1-C18烷基、C2-C18烯基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7、(C1-C4烷基)(C3-C9杂芳基)和C1-C12烷基(W)C1-C12烷基，其中W为选自N、S和O的杂原子，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基或芳基；或者R4和R8与它们所连接的原子一起形成C3-C6环烷基；R1 , R2 , R4 and R8 are independently selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C1 -C18 alkyl)OH, (C1 -C18 alkane base) SH, (C2 -C3 alkyl) SCH3 , (C1 -C4 alkyl) CONH2 , (C1 -C4 alkyl) COOH, (C1 -C4 alkyl) NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 , (C1 -C4 alkyl) (C3 -C9 heteroaryl) and C1 -C12 alkyl (W) C1 -C12 alkyl, wherein W is a heteroatom selected from N, S and O, or R1 and R2 together with the atoms to which they are attached form C3 -C12 cycloalkyl or aryl; or R4 and R8 together with the atoms they are connected to form a C3 -C6 cycloalkyl;
R3选自C1-C18烷基、(C1-C18烷基)OH、(C1-C18烷基)NH2、(C1-C18烷基)SH、(C0-C4烷基)(C3-C6)环烷基、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和(C1-C4烷基)(C3-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成4、5或6元杂环；R3 is selected from C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C18 alkyl) NH2 , (C1 -C18 alkyl) SH, (C0 - C4 alkyl) (C3 -C6 ) cycloalkyl, (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and (C1 -C4 alkyl) (C3 -C9 heteroaryl), or R4 and R3 form a 4, 5 or 6 membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R2与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R2 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自H和OH，前提条件是如果R4和R3与它们所连接的原子一起形成4、5或6元杂环，则R1和R2两者都不为H。R7 is selected from H and OH, with the proviso that neither R1 nor R2 is H if R4 andR3 together with the atoms to which they are attached forma4 ,5 or 6 membered heterocyclic ring.
在另一个实施方案中，二肽前药元件包含以下通用结构：In another embodiment, the dipeptide prodrug element comprises the following general structure:
其中in
R1和R8独立地为H或C1-C8烷基；R1 and R8 are independently H or C1 -C8 alkyl;
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C3-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclic radical), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and CH2 (C3 -C9 heteroaryl), or R1 and R2 together with the atoms to which they are attached Formation of a C3 -C12 cycloalkyl group;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)NH2、(C1-C4烷基)SH、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) NH2 , (C1 -C4 alkyl) SH, (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R2 together with the atoms to which they are attached form a 5- or 6-membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素，前提条件是如果式I的二肽通过某一肽的N端胺连接，且R4和R3与它们所连接的原子一起形成5或6元杂环，则R1和R2两者都不为H。在一个实施方案中，二肽前药元件的第一氨基酸和/或第二氨基酸为呈D立体异构体构型的氨基酸。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl)NH2 , (C0 -C4 alkyl)OH and halogen, provided that if the dipeptide of formula I is attached via the N-terminal amine of a peptide, and R4 and R3 are connected to the atoms together form a 5- or6- membered heterocyclic ring, neither R nor R is H. In one embodiment, the first amino acid and/or the second amino acid of the dipeptide prodrug element is an amino acid in the D stereoisomer configuration.
在一个实施方案中，提供式I的前药元件，其中R1选自H和C1-C8烷基；和In one embodiment, there is provideda prodrug element of formula I, wherein R is selected from H and C 1-C8 alkyl; and
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C8环烷基环；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 and CH2 (C5 -C9 heteroaryl), or R1 and R2 together with the atoms they are connected to form a C3 -C8 cycloalkyl ring;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；R6 is H, or R6 and R2 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R7选自H和OH，R8为H。在一个实施方案中，R3为C1-C8烷基，R4选自H、C1-C6烷基、CH2OH、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成5或6元杂环。在另一个的实施方案中，R5为NHR6，R8为H。R7 is selected from H and OH,R8 is H. In one embodiment, R3 is C1 -C8 alkyl, R4 is selected from H, C1 -C6 alkyl, CH2 OH, (C0 -C4 alkyl) (C6 -C10 Aryl) R7 and CH2 (C5 -C9 heteroaryl), or R4 and R3 together with the atoms they are attached to form a 5- or 6-membered heterocyclic ring.In another embodiment,R5 is NHR6 andR8 is H.
在另一个实施方案中，提供式I的前药元件，其中In another embodiment, there is provided a prodrug element of formula I, wherein
R1选自H、C1-C8烷基；和R1 is selected from H, C1 -C8 alkyl; and
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C8环烷基环；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 and CH2 (C5 -C9 heteroaryl), or R1 and R2 together with the atoms they are connected to form a C3 -C8 cycloalkyl ring;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；R6 is H, or R6 and R2 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素；R8为H，前提条件是如果R4和R3与它们所连接的原子一起形成5或6元杂环，则R1和R2两者都不为H。在一个实施方案中，二肽前药元件的第一氨基酸和/或第二氨基酸为非编码氨基酸，在一个实施方案中为呈D立体异构体构型的氨基酸。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen;R8 is H, with the proviso that if R4 andR3 together with the atoms to which they are attached form a 5- or6 -membered heterocyclic ring, thenNeitherR1 nor R2 is H. In one embodiment, the first amino acid and/or the second amino acid of the dipeptide prodrug element is a non-coded amino acid, in one embodiment an amino acid in the D stereoisomer configuration.
在其它实施方案中，二肽前药元件具有式I的结构，其中In other embodiments, the dipeptide prodrug element has the structure of Formula I, wherein
R1和R8独立地为H或C1-C8烷基；R1 and R8 are independently H or C1 -C8 alkyl;
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C3-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2 +) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclic radical), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and CH2 (C3 -C9 heteroaryl), or R1 and R2 together with the atoms to which they are attached Formation of a C3 -C12 cycloalkyl group;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R5为NHR6；R5 is NHR6 ;
R6为H或C1-C8烷基；R6 is H or C1 -C8 alkyl;
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素，R8为H。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl) NH2 , (C0 -C4 alkyl) OH and halogen, R8 is H.
在另一个实施方案中，二肽前药元件具有式I的结构，其中In another embodiment, the dipeptide prodrug element has the structure of Formula I, wherein
R1和R2独立地为C1-C18烷基或(C0-C4烷基)(C6-C10芳基)R7；或R1和R2通过-(CH2)p连接，其中p为2-9；R1 and R2 are independently C1 -C18 alkyl or (C0 -C4 alkyl) (C6 -C10 aryl) R7 ; or R1 and R2 pass through -(CH2 )p Connect, where p is 2-9;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R4和R8各自为氢；R and Rare each hydrogen;
R5为NH2；和R5 is NH2 ; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
在另一个实施方案中，二肽前药元件具有式I的结构，其中In another embodiment, the dipeptide prodrug element has the structure of Formula I, wherein
R1和R2独立选自氢、C1-C18烷基、(C1-C18烷基)OH、(C1-C4烷基)NH2和(C0-C4烷基)(C6-C10芳基)R7，或者R1和R2通过(CH2)p连接，其中p为2-9；R1 and R2 are independently selected from hydrogen, C1 -C18 alkyl, (C1 -C18 alkyl)OH, (C1 -C4 alkyl)NH2 and (C0 -C4 alkyl) (C6 -C10 aryl) R7 , or R1 and R2 are linked by (CH2 )p , where p is 2-9;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-12元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-12 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立选自氢、C1-C8烷基和(C0-C4烷基)(C6-C10芳基)R7；R4 and R8 are independently selected from hydrogen, C1 -C8 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R5为NH2；和R5 is NH2 ; and
R7选自H、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素，前提条件是R1和R2两者都不为氢，且前提条件是R4或R8中的至少一个为氢。R7 is selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl)NH2 , (C0 -C4 alkyl)OH and halogen, with the proviso that neither R nor R ishydrogen , and with theproviso that at leastone ofR or R is hydrogen .
在另一个实施方案中，二肽前药元件具有式I的结构，其中In another embodiment, the dipeptide prodrug element has the structure of Formula I, wherein
R1和R2独立选自氢、C1-C8烷基和(C1-C4烷基)NH2，或者R1和R2通过(CH2)p连接，其中p为2-9；R1 and R2 are independently selected from hydrogen, C1 -C8 alkyl and (C1 -C4 alkyl)NH2 , or R1 and R2 are linked by (CH2 )p , where p is 2-9 ;
R3为C1-C8烷基，或者R3和R4与它们所连接的原子一起形成4-6元杂环；R3 is a C1 -C8 alkyl group, or R3 and R4 form a 4-6 membered heterocyclic ring together with the atoms they are connected to;
R4选自氢和C1-C8烷基；R4 is selected from hydrogen and C1 -C8 alkyl;
R8为氢；和R is hydrogen; and
R5为NH2，前提条件是R1和R2两者都不为氢。R5 is NH2 with the proviso that neither R1 nor R2 is hydrogen.
在另一个实施方案中，二肽前药元件具有式I的结构，其中In another embodiment, the dipeptide prodrug element has the structure of Formula I, wherein
R1和R2独立选自氢、C1-C8烷基和(C1-C4烷基)NH2；R1 and R2 are independently selected from hydrogen, C1 -C8 alkyl and (C1 -C4 alkyl) NH2 ;
R3为C1-C6烷基；R3 is C1 -C6 alkyl;
R4和R8各自为氢；和R and Rare each hydrogen; and
R5为NH2，前提条件是R1和R2两者都不为氢。R5 is NH2 with the proviso that neither R1 nor R2 is hydrogen.
在另一个实施方案中，二肽前药元件具有式I的结构，其中In another embodiment, the dipeptide prodrug element has the structure of Formula I, wherein
R1和R2独立选自氢和C1-C8烷基、(C1-C4烷基)NH2，或者R1和R2通过(CH2)p连接，其中p为2-9；R1 and R2 are independently selected from hydrogen and C1 -C8 alkyl, (C1 -C4 alkyl)NH2 , or R1 and R2 are linked by (CH2 )p , where p is 2-9 ;
R3为C1-C8烷基；R3 is C1 -C8 alkyl;
R4为(C0-C4烷基)(C6-C10芳基)R7；R4 is (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R5为NH2；R5 is NH2 ;
R7选自氢、C1-C8烷基和(C0-C4烷基)OH；和R7 is selected from hydrogen, C1 -C8 alkyl and (C0 -C4 alkyl)OH; and
R8为氢，前提条件是R1和R2两者都不为氢。R8 is hydrogen with the proviso that neitherR1 nor R2 ishydrogen .
在另一个实施方案中，二肽前药元件具有式I的结构，其中In another embodiment, the dipeptide prodrug element has the structure of Formula I, wherein
R1选自氢、C1-C8烷基和(C0-C4烷基)(C6-C10芳基)R7；R1 is selected from hydrogen, C1 -C8 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R2为氢；R ishydrogen ;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R4和R8各自为氢；R and Rare each hydrogen;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素，前提条件是，如果R1为烷基或(C0-C4烷基)(C6-C10芳基)R7，则R1和R5与它们所连接的原子一起形成4-11元杂环。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl)NH2 , (C0 -C4 alkyl)OH and halogen, with the proviso that if R1 is alkyl or (C0 -C4 alkyl)(C6 -C10 aryl)R7 , then R1 and R5 form a 4-11 membered heterocyclic ring together with the atoms they are connected to.
根据一个实施方案，提供包含胰岛素肽和酰胺连接的二肽的胰岛素前药类似物。更具体地讲，胰岛素前药类似物包含A链序列和B链序列，其中A链包含序列Z-GIVEQCCX1SICSLYQLENX2CX3-R13(SEQ ID NO：3)或者其类似物，该类似物包含因1-9、1-5或1-3个选自以下位置的氨基酸修饰而不同于SEQ ID NO：3的序列：A5、A8、A9、A10、A14、A15、A17、A18(相对于天然胰岛素A链)，B链序列包含序列J-X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)或者其类似物，该类似物包含因1-10、1-5或1-3个选自以下位置的氨基酸修饰而不同于SEQ ID NO：14序列的序列：B1、B2、B3、B4、B5、B13、B14、B17、B20、B22、B23、B26、B27、B28、B29和B30(相对于天然胰岛素B链；即SEQ ID NO：14的氨基酸X4相当于天然胰岛素的B5位)。Z和J独立地为H或包含下式I的通用结构的二肽；According to one embodiment, there is provided an insulin prodrug analog comprising an insulin peptide and an amide-linked dipeptide. More specifically, the insulin prodrug analogue comprises an A-chain sequence and a B-chain sequence, wherein the A-chain comprises the sequence Z-GIVEQCCX1 SICSLYQLENX2 CX3 -R13 (SEQ ID NO: 3) or an analogue thereof, the analogue Comprising a sequence different from SEQ ID NO: 3 due to 1-9, 1-5 or 1-3 amino acid modifications selected from the following positions: A5, A8, A9, A10, A14, A15, A17, A18 (relative to natural insulin A chain), the sequence of the B chain comprises the sequence JX14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14) or its analogues, the analogues comprising factors 1-10, 1-5 or 1 -3 amino acid modifications selected from the following positions differing from the sequence of SEQ ID NO: 14: B1, B2, B3, B4, B5, B13, B14, B17, B20, B22, B23, B26, B27, B28, B29 and B30 (relative to native insulin B chain; ie amino acid X4 of SEQ ID NO: 14 corresponds to position B5 of native insulin). Z and J are independently H or a dipeptide comprising the general structure of Formula I below;
X14或为将“J”元件与X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)序列连接的键，或者X14表示选自以下的将“J”元件与X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)序列连接的1-4个氨基酸的序列：X9VNQ(SEQ ID NO：21)、VNQ、NQ和Q。X14 is either a bond linking the "J" element to X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14) sequence, or X14 represents a combination of the "J" element and X4 LCGX5 selected from the following A sequence of 1-4 amino acids linked by X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14): X9 VNQ (SEQ ID NO: 21), VNQ, NQ and Q.
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中m为选自0-3的整数，X选自OH、NH2、NHR10和OCH3，其中R10为H或包含下式I的通用结构的二肽：wherein m is an integer selected from 0-3, X is selected from OH, NH2 , NHR10 and OCH3 , wherein R10 is H or a dipeptide comprising the general structure of formula I below:
X3选自天冬酰胺、甘氨酸、丙氨酸、苏氨酸和丝氨酸；X is selected fromasparagine , glycine, alanine, threonine and serine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X7为以下通用结构的氨基酸X7 is an amino acid of the following general structure
其中m为选自0-3的整数，X12选自OH、NH2、NHR11和OCH3，其中R11为H或包含下式I的通用结构的二肽：Wherein m is an integer selected from 0-3, X12 is selected from OH, NH2 , NHR11 and OCH3 , wherein R11 is H or a dipeptide comprising the general structure of the following formula I:
X8为以下通用结构的氨基酸X8 is an amino acid of the following general structure
其中m为选自0-3的整数，X13选自H、OH、NH2、NHR12和OCH3，其中R12为H或包含下式I的通用结构的二肽：wherein m is an integer selected from 0-3, X13 is selected from H, OH, NH2 , NHR12 and OCH3 , wherein R12 is H or a dipeptide comprising the general structure of the following formula I:
X9选自苯丙氨酸和脱氨基-苯丙氨酸；其中X is selected from phenylalanine anddesamino -phenylalanine; wherein
R1、R2、R4和R8独立选自H、C1-C18烷基、C2-C18烯基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7、(C1-C4烷基)(C3-C9杂芳基)和C1-C12烷基(W)C1-C12烷基，其中W为选自N、S和O的杂原子，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基或芳基；或者R4和R8与它们所连接的原子一起形成C3-C6环烷基；R1 , R2 , R4 and R8 are independently selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C1 -C18 alkyl)OH, (C1 -C18 alkane base) SH, (C2 -C3 alkyl) SCH3 , (C1 -C4 alkyl) CONH2 , (C1 -C4 alkyl) COOH, (C1 -C4 alkyl) NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 , (C1 -C4 alkyl) (C3 -C9 heteroaryl) and C1 -C12 alkyl (W) C1 -C12 alkyl, wherein W is a heteroatom selected from N, S and O, or R1 and R2 together with the atoms to which they are attached form C3 -C12 cycloalkyl or aryl; or R4 and R8 together with the atoms they are connected to form a C3 -C6 cycloalkyl;
R3选自C1-C18烷基、(C1-C18烷基)OH、(C1-C18烷基)NH2、(C1-C18烷基)SH、(C0-C4烷基)(C3-C6)环烷基、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和(C1-C4烷基)(C3-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成4、5或6元杂环；R3 is selected from C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C18 alkyl) NH2 , (C1 -C18 alkyl) SH, (C0 - C4 alkyl) (C3 -C6 ) cycloalkyl, (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and (C1 -C4 alkyl) (C3 -C9 heteroaryl), or R4 and R3 form a 4, 5 or 6 membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R2与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R2 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自H和OH；和Ris selected from H and OH; and
R13为COOH或CONH2。在一个实施方案中，R13为COOH，B链的羧基端氨基酸具有替代天然α碳羧基的酰胺(CONH2)。在一个实施方案中，X、X12、X13、J和Z中的一个或多个为包含下式I通用结构的二肽：R13 is COOH or CONH2 . In one embodiment,R13 is COOH and the carboxy-terminal amino acid of the B chain has an amide (CONH2) in place of the natural alpha carbon carboxyl group. In one embodiment, one or more of X,X12 ,X13 , J and Z is a dipeptide comprising the general structure of Formula I below:
而在一个实施方案中，X、X12、X13、J和Z中的两个包含下式I通用结构的二肽： While in one embodiment, two of X, X12 , X13 , J and Z comprise a dipeptide of the general structure of Formula I below:
根据一个实施方案，X、X12、X13、J和Z中的至少一个为包含下式I通用结构的二肽： According to one embodiment, at least one of X,X12 ,X13 , J and Z is a dipeptide comprising the general structure of formula I below:
而在一个实施方案中，X、X12、X13、J和Z中的一个并且只有一个包含下式I通用结构的二肽： Yet in one embodiment, one and only one of X, X12 , X13 , J and Z comprises a dipeptide having the general structure of Formula I below:
(即只有一个二肽前药元件与胰岛素肽连接)。此外，如果二肽前药元件与A链或B链的N端连接(即J或Z中的任一个包含二肽)，且R4和R3与它们所连接的原子一起形成4、5或6元杂环，则R1和R2中的至少一个不为H，而在一个实施方案中，R1和R2两者都不为H。在一个实施方案中，J和Z两者都为H，X12为OH，X13为H或OH，X为NHR10，其中R10为包含式I通用结构的二肽。在另一个实施方案中，A链包含序列GIVEQCCX1SICSLYQLENX2CX3-R13(SEQ ID NO：3)，B链序列包含序列X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)，m为1，X12为OH，X13为H或OH，X为NHR10，其中R10为包含式I通用结构的二肽，R13为COOH，B链的羧基端氨基酸具有替代天然α碳羧基的酰胺(CONH2)，其余标记如紧接的上文中所定义。 (ie only one dipeptide prodrug element is linked to the insulin peptide). In addition, if the dipeptide prodrug element is attached to the N-terminus of either chain A or B (i.e. either J or Z comprises a dipeptide) and R4 andR3 together with the atoms to which they are attached form4 , 5 or6- membered heterocycle, then at leastone of R and R is not H, and inone embodiment neither R nor R is H. In one embodiment, both J and Z are H, X12 is OH, X13 is H or OH, X is NHR10 , wherein R10 is a dipeptide comprising the general structure of Formula I. In another embodiment, the A chain comprises the sequence GIVEQCCX1 SICSLYQLENX2 CX3 -R13 (SEQ ID NO: 3), and the B chain sequence comprises the sequence X14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO : 14), m is 1, X12 is OH, X13 is H or OH, X is NHR10 , wherein R10 is a dipeptide comprising the general structure of formula I, R13 is COOH, and the carboxy-terminal amino acid of the B chain has Amide (CONH2 ) substituting the natural alpha carbon carboxyl, the rest of the labels are as defined immediately above.
在一个实施方案中，存在于X、X12、X13、J和Z上的二肽为包含下式I通用结构的二肽：In one embodiment, the dipeptides present at X,X12 ,X13 , J and Z are dipeptides comprising the general structure of Formula I below:
其中in
R1、R2、R4和R8独立选自H、C1-C18烷基、C2-C18烯基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7、(C1-C4烷基)(C3-C9杂芳基)和C1-C12烷基(W1)C1-C12烷基，其中W1为选自N、S和O的杂原子，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基；或者R4和R8与它们所连接的原子一起形成C3-C6环烷基；R1 , R2 , R4 and R8 are independently selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C1 -C18 alkyl)OH, (C1 -C18 alkane base) SH, (C2 -C3 alkyl) SCH3 , (C1 -C4 alkyl) CONH2 , (C1 -C4 alkyl) COOH, (C1 -C4 alkyl) NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 , (C1 -C4 alkyl) (C3 -C9 heteroaryl) and C1 -C12 alkyl (W1 )C1 -C12 alkyl, wherein W1 is a heteroatom selected from N, S and O, or R1 and R2 together with the atoms to which they are attached form C3 -C12 cycloalkyl; or R4 and R8 form C3 -C6 cycloalkyl together with the atoms they are connected to;
R3选自C1-C18烷基、(C1-C18烷基)OH、(C1-C18烷基)NH2、(C1-C18烷基)SH、(C0-C4烷基)(C3-C6)环烷基、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和(C1-C4烷基)(C3-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成4、5或6元杂环；R3 is selected from C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C18 alkyl) NH2 , (C1 -C18 alkyl) SH, (C0 - C4 alkyl) (C3 -C6 ) cycloalkyl, (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and (C1 -C4 alkyl) (C3 -C9 heteroaryl), or R4 and R3 form a 4, 5 or 6 membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素，前提条件是如果R4和R3与它们所连接的原子一起形成5或6元杂环，则R1和R2两者都不为H。在一个实施方案中，如果J或Z包含式I的二肽，且R4和R3与它们所连接的原子一起形成4、5或6元杂环，则R1和R2两者都不为氢。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen, with the proviso that if R4 andR3 together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring, then R1 andR2 Neither is H. In one embodiment, if J or Z comprises a dipeptide of formula I, and R andR together with the atoms to which they are attached form a4 ,5 or6 membered heterocyclic ring, neither R nor R for hydrogen.
在一个实施方案中，R1选自H和C1-C8烷基；In one embodiment, R1 is selected from H and C1 -C8 alkyl;
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 and CH2 (C5 -C9 heteroaryl);
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；R6 is H, or R6 and R2 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R7选自H和OH，R8为H。根据另一个实施方案，m为1，R8为H，R3为C1-C6烷基，R4选自H、C1-C4烷基、(C3-C6)环烷基、(C1-C4烷基)OH、(C1-C4烷基)SH和(C0-C4烷基)(C6芳基)R7，或者R3和R4与它们所连接的原子一起形成5元杂环。在一个实施方案中，m为1，R8为H，R3为C1-C6烷基，R4选自H、C1-C4烷基，或者R3和R4与它们所连接的原子一起形成5元杂环。在另一个实施方案中，X9为苯丙氨酸，且单个二肽延伸物通过A链或B链N端的酰胺键与胰岛素肽连接。在一个备选实施方案中，胰岛素肽包含在A链19位上的4氨基苯丙氨酸取代，且单个二肽延伸物通过在4氨基苯丙氨酸的芳族胺上形成的酰胺键与胰岛素肽连接。在一个实施方案中，本文公开的胰岛素类似物包含C端酰胺或酯，替换A链和/或B链的C端羧酸酯。R7 is selected from H and OH,R8 is H. According to another embodiment, m is 1, R8 is H, R3 is C1 -C6 alkyl, R4 is selected from H, C1 -C4 alkyl, (C3 -C6 )cycloalkyl , (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH and (C0 -C4 alkyl) (C6 aryl) R7 , or R3 and R4 with their The linked atoms are taken together to form a 5-membered heterocyclic ring. In one embodiment, m is 1, R8 is H, R3 is C1 -C6 alkyl, R4 is selected from H, C1 -C4 alkyl, or R3 and R4 are connected to The atoms together form a 5-membered heterocyclic ring. In another embodiment,X9 is phenylalanine, and a single dipeptide extension is linked to the insulin peptide via an amide bond at the N-terminus of the A or B chains. In an alternative embodiment, the insulin peptide comprises a 4-aminophenylalanine substitution at position 19 of the A chain, and a single dipeptide extension is linked to Insulin peptide linkage. In one embodiment, an insulin analog disclosed herein comprises a C-terminal amide or ester, replacing the C-terminal carboxylate of the A chain and/or B chain.
根据一个实施方案，将式I的二肽进一步修饰以包含大的聚合物，其干扰胰岛素类似物与胰岛素或IGF-1受体相互作用的能力。二肽随后裂解从二肽复合体中释放出胰岛素类似物，其中释放的胰岛素类似物具有完整活性。根据一个实施方案，J包含式I的二肽，其中将式I的二肽进一步修饰以包含大的聚合物，其干扰结合的胰岛素类似物与胰岛素或IGF-1受体相互作用的能力。根据一个实施方案，X、X12、X13、J和Z之一包含下式I通用结构的二肽：According to one embodiment, the dipeptide of formula I is further modified to comprise bulky polymers which interfere with the ability of the insulin analog to interact with insulin or the IGF-1 receptor. Subsequent cleavage of the dipeptide releases the insulin analog from the dipeptide complex, wherein the released insulin analog is fully active. According to one embodiment, J comprises a dipeptide of formula I, wherein the dipeptide of formula I is further modified to comprise bulky polymers which interfere with the ability of the bound insulin analog to interact with insulin or the IGF-1 receptor. According to one embodiment, one of X, X12 , X13 , J and Z comprises a dipeptide of the general structure of formula I below:
其中将式I的二肽聚乙二醇化、烷基化或酰化。在一个实施方案中，J、Z或X中的任一个包含式I的酰化或聚乙二醇化二肽，而在一个实施方案中，J包含式I的酰化或聚乙二醇化二肽。 wherein the dipeptide of formula I is pegylated, alkylated or acylated. In one embodiment, any of J, Z or X comprises an acylated or PEGylated dipeptide of Formula I, and in one embodiment J comprises an acylated or PEGylated dipeptide of Formula I .
在一个实施方案中，二肽前药元件在A链或B链的N端通过酰胺键与胰岛素肽共价结合，或者与携带内部氨基酸侧链的胺连接，其中二肽还包含与二肽连接的长效聚合物。在一个实施方案中，胰岛素肽的天然氨基酸被适于与式I的二肽形成酰胺键的氨基酸取代。在一个实施方案中，携带二肽的长效物在选自以下的位置上连接：A14、A19、B16、B28和B29。在一个实施方案中，两个或更多个长效聚合物与单个二肽元件连接。将长效聚合物经选择以具有生物相容性和足够大小，使得通过共价连接二肽修饰的胰岛素肽在给予患者时在注射部位保持螯合和/或不能够与其相应受体相互作用。二肽随后裂解释放出胰岛素肽以与预期靶标相互作用。In one embodiment, the dipeptide prodrug element is covalently bound to the insulin peptide via an amide bond at the N-terminus of the A or B chain, or linked to an amine bearing an internal amino acid side chain, wherein the dipeptide further comprises a link to the dipeptide. long-lasting polymers. In one embodiment, the natural amino acid of the insulin peptide is substituted with an amino acid suitable for forming an amide bond with the dipeptide of formula I. In one embodiment, the dipeptide-bearing depot is attached at a position selected from: A14, A19, B16, B28 and B29. In one embodiment, two or more long-lasting polymers are linked to a single dipeptide element. The long-acting polymer is selected to be biocompatible and of sufficient size such that the insulin peptide modified by covalently linking the dipeptide remains sequestered at the injection site and/or is unable to interact with its corresponding receptor when administered to a patient. The dipeptide is then cleaved to release the insulin peptide to interact with the intended target.
根据一个实施方案，长效聚合物选自本领域技术人员已知的生物相容性聚合物。长效聚合物的大小通常约为20,000-120,000道尔顿。在一个实施方案中，长效聚合物的大小为约40,000-100,000或约40,000-80,000道尔顿。在一个实施方案中，长效聚合物的大小为约40,000、50,000、60,000、70,000或80,000道尔顿。合适的长效聚合物包括但不限于葡聚糖、聚丙交酯、聚乙交酯、己内酯型聚合物、聚己内酯、聚酐、聚胺、聚酰胺酯、聚原酸酯、聚二氧杂环己酮、聚缩醛、聚缩酮、聚碳酸酯、聚磷酸酯、聚酯、聚对苯二甲酸丁二酯、聚原碳酸酯、聚磷腈、琥珀酸酯、聚苹果酸、聚氨基酸、聚乙烯吡咯烷酮、聚乙二醇、聚羟基纤维素、多糖、壳多糖、脱乙酰壳多糖、透明质酸及其共聚物、三元共聚物和混合物，以及生物可降解聚合物及其共聚物，包括己内酯型聚合物、聚己内酯和共聚物(其包括聚对苯二甲酸丁二酯)。在一个实施方案中，长效聚合物选自聚乙二醇、葡聚糖、聚乳酸、聚乙醇酸和乳酸与乙醇酸的共聚物，而在一个具体实施方案中，长效聚合物为聚乙二醇。在一个实施方案中，长效聚合物为聚乙二醇，且与二肽元件连接的长效聚合物总的分子量约为40,000-80,000道尔顿。According to one embodiment, the long-acting polymer is selected from biocompatible polymers known to those skilled in the art. Long-lasting polymers are typically about 20,000-120,000 Daltons in size. In one embodiment, the long-lived polymer has a size of about 40,000-100,000 or about 40,000-80,000 Daltons. In one embodiment, the long-lived polymer has a size of about 40,000, 50,000, 60,000, 70,000, or 80,000 Daltons. Suitable long-lasting polymers include, but are not limited to, dextran, polylactide, polyglycolide, caprolactone-type polymers, polycaprolactone, polyanhydrides, polyamines, polyesteramides, polyorthoesters, Polydioxanone, polyacetal, polyketal, polycarbonate, polyphosphate, polyester, polybutylene terephthalate, polyorthocarbonate, polyphosphazene, succinate, poly Malic acid, polyamino acids, polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, polysaccharides, chitin, chitosan, hyaluronic acid and its copolymers, terpolymers and blends, and biodegradable polymers and copolymers thereof, including caprolactone-type polymers, polycaprolactone and copolymers including polybutylene terephthalate. In one embodiment, the long-acting polymer is selected from the group consisting of polyethylene glycol, dextran, polylactic acid, polyglycolic acid, and copolymers of lactic and glycolic acid, and in a specific embodiment, the long-acting polymer is poly ethylene glycol. In one embodiment, the long-acting polymer is polyethylene glycol, and the total molecular weight of the long-acting polymer attached to the dipeptide element is about 40,000-80,000 Daltons.
根据一个实施方案，式I的二肽还包含聚环氧乙烷、烷基或酰基。在一个实施方案中，一条或多条聚环氧乙烷链与式I的二肽连接，其中聚环氧乙烷链总的分子量为约20,000-约80,000道尔顿，或40,000-80,000道尔顿或40,000-60,000道尔顿。在一个实施方案中，聚环氧乙烷为聚乙二醇。在一个实施方案中，至少一条分子量约40,000道尔顿的聚乙二醇链与式I的二肽连接。在另一个实施方案中，将式I的二肽用具有足够大小的酰基酰化以结合血清白蛋白，从而在给药时使胰岛素类似物失活。酰基可以是直链或支链的，而在一个实施方案中是C16-C30脂肪酸。例如，酰基可以是C16脂肪酸、C18脂肪酸、C20脂肪酸、C22脂肪酸、C24脂肪酸、C26脂肪酸、C28脂肪酸或C30脂肪酸中的任一种。在一个实施方案中，酰基是C16-C20脂肪酸，例如C18脂肪酸或C20脂肪酸。According to one embodiment, the dipeptide of formula I further comprises a polyethylene oxide, an alkyl or an acyl group. In one embodiment, one or more polyethylene oxide chains are attached to the dipeptide of formula I, wherein the total molecular weight of the polyethylene oxide chains is from about 20,000 to about 80,000 Daltons, or from 40,000 to 80,000 Daltons Ton or 40,000-60,000 Dalton. In one embodiment, the polyethylene oxide is polyethylene glycol. In one embodiment, at least one polyethylene glycol chain having a molecular weight of about 40,000 Daltons is linked to the dipeptide of Formula I. In another embodiment, the dipeptide of formula I is acylated with an acyl group of sufficient size to bind serum albumin, thereby inactivating the insulin analog upon administration. The acyl group can be straight or branched and in one embodiment is a C16-C30 fatty acid. For example, the acyl group can be any of a C16 fatty acid, a C18 fatty acid, a C20 fatty acid, a C22 fatty acid, a C24 fatty acid, a C26 fatty acid, a C28 fatty acid, or a C30 fatty acid. In one embodiment, the acyl group is a C16-C20 fatty acid, such as a C18 fatty acid or a C20 fatty acid.
根据一个实施方案，提供提供包含A链序列和B链序列的胰岛素前药类似物，其中A链包含序列GIVEQCCX1SICSLYQLENX2CX3-R13(SEQ ID NO：3)，B链序列包含序列X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)，其中According to one embodiment, there is provided an insulin prodrug analogue comprising an A chain sequence and a B chain sequence, wherein the A chain comprises the sequence GIVEQCCX1 SICSLYQLENX2 CX3 -R13 (SEQ ID NO: 3), and the B chain sequence comprises the sequence X14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14), wherein
X14选自将“J”元件与X4LCGX5X6LVEALX7LVCGERGFX8-R14(SEQ ID NO：14)序列连接的N端胺、X9VNQ(SEQ ID NO：21)、VNQ、NQ和Q。X14 is selected from the group consisting of the N-terminal amine linking the "J" element to the sequence X4 LCGX5 X6 LVEALX7 LVCGERGFX8 -R14 (SEQ ID NO: 14), X9 VNQ (SEQ ID NO: 21 ), VNQ, NQ and Q.
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中U为氨基酸或羟酸，O为通过酰胺键连接的N-烷基化氨基酸；Wherein U is an amino acid or a hydroxy acid, and O is an N-alkylated amino acid linked by an amide bond;
X3选自天冬酰胺、鸟氨酸、甘氨酸、丙氨酸、苏氨酸和丝氨酸；X is selected from asparagine,ornithine , glycine, alanine, threonine and serine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X7为以下通用结构的氨基酸X7 is an amino acid of the following general structure
其中m为选自0-3的整数，X12选自OH、NH2和OCH3；Wherein m is an integer selected from 0-3, X12 is selected from OH, NH2 and OCH3 ;
X8为以下通用结构的氨基酸X8 is an amino acid of the following general structure
其中m为选自0-3的整数，X13选自H、OH、NH2和OCH3；Wherein m is an integer selected from 0-3, X13 is selected from H, OH, NH2 and OCH3 ;
X9选自苯丙氨酸和脱氨基-苯丙氨酸；R13和R14独立地为COOH或CONH2。在一个实施方案中，X7和X8两者都为酪氨酸，R13为COOH，B链的羧基端氨基酸具有替代天然α碳羧基的酰胺(CONH2)。X9 is selected from phenylalanine and desamino-phenylalanine; R13 and R14 are independently COOH or CONH2 .In one embodiment, both X7 andX8 are tyrosine,R13 is COOH, and the carboxy-terminal amino acid of the B chain has an amide (CONH2) replacing the natural alpha-carbon carboxyl group.
根据一个实施方案，提供包含序列Z-GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)或其类似物的化合物，该类似物包含因1-3个选自以下位置的氨基酸修饰而不同于SEQ IDNO：3的序列：A5、A8、A9、A10、A14、A15、A17、A18(相对于天然胰岛素A链序列)。在这个实施方案中，X1选自苏氨酸和组氨酸；According to one embodiment, there is provided a compound comprising the sequence Z-GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) or an analogue thereof which differs from SEQ ID NO: 3 by 1-3 amino acid modifications selected from the following positions: Sequence of ID NO: 3: A5, A8, A9, A10, A14, A15, A17, A18 (relative to the native insulin A chain sequence).In this embodiment, X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NH2、NHR10和OCH3，wherein X is selected from OH, NH2 , NHR10 and OCH3 ,
而且其中R10和Z独立地为H或结构U-O的二肽，其中U为氨基酸或羟酸，O为N-烷基化氨基酸，其中O通过形成酰胺键与SEQ ID NO：3的肽连接，前提条件是R10和Z不同，且U、O或U-O与之连接的SEQ ID NO：3的氨基酸为非编码氨基酸。在一个实施方案中，在生理条件下，在PBS中，二肽从SEQ ID NO：3中的化学裂解在约1小时-约720小时内完成至少约90％。在另一个实施方案中，在生理条件下，在PBS中，U-O自SEQ ID NO：3的化学裂解半寿期(t1/2)为至少约1小时-约1周。在一个实施方案中，化合物还包含通过分子间二硫键或作为重组单链多肽与SEQ ID NO：3的A链连接的胰岛素B链。And whereinR and Z are independently H or a dipeptide of structure UO, wherein U is an amino acid or a hydroxyacid, O is an N-alkylated amino acid, wherein O is linked to the peptide of SEQ ID NO: 3 by forming an amide bond, The prerequisite is that R10 and Z are different, and the amino acid of SEQ ID NO: 3 to which U, O or UO is linked is a non-coding amino acid. In one embodiment, chemical cleavage of the dipeptide from SEQ ID NO: 3 is at least about 90% complete within about 1 hour to about 720 hours in PBS under physiological conditions. In another embodiment, UO has a chemical cleavage half-life (t1/2 ) from SEQ ID NO: 3 in PBS of at least about 1 hour to about 1 week under physiological conditions. In one embodiment, the compound further comprises an insulin B chain linked to the A chain of SEQ ID NO: 3 by an intermolecular disulfide bond or as a recombinant single chain polypeptide.
二肽元件上的取代基的选择和二肽前药元件的连接位点可影响二肽前药元件从胰岛素肽上进行化学裂解的速率。在一个实施方案中，提供包含A链序列和B链序列的胰岛素前药，其中A链包含序列GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)，B链序列包含序列X14-X4LCGX5X6LVEALYLVCGERGFF(SEQ ID NO：4)，其中The choice of substituents on the dipeptide element and the attachment site of the dipeptide prodrug element can affect the rate at which the dipeptide prodrug element undergoes chemical cleavage from the insulin peptide. In one embodiment, there is provided an insulin prodrug comprising an A chain sequence comprising the sequence GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) and a B chain sequence comprising the sequence X14 -X4 LCGX5 X6 LVEALYLVCGERGFF (SEQ ID NO: 4), wherein
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NH2和OCH3；Wherein X is selected from OH, NH2 and OCH3 ;
X3选自天冬酰胺、甘氨酸、丙氨酸、苏氨酸和丝氨酸；X is selected fromasparagine , glycine, alanine, threonine and serine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；和X is selected from histidine, aspartic acid, glutamic acid, homocysteine and cysteine; and
X14选自键、X9VNQ(SEQ ID NO：21)、VNQ、NQ和Q，X9选自苯丙氨酸和脱氨基-苯丙氨酸，而且其中包含以下结构的二肽前药元件与肽SEQ ID NO：3或SEQ ID NO：4的N端氨基酸的α氨基连接：X14 is selected from bond, X9 VNQ (SEQ ID NO: 21), VNQ, NQ and Q, X9 is selected from phenylalanine and deamino-phenylalanine, and wherein comprises a dipeptide prodrug of the following structure The element is linked to the alpha amino group of the N-terminal amino acid of peptide SEQ ID NO: 3 or SEQ ID NO: 4:
前提条件是如果R4和R3与它们所连接的原子一起形成4、5或6元杂环，则R1和R2两者都不为氢。在这个实施方案中，提供在生理条件下，在PBS中t1/2为约1小时的化合物，其中The proviso is that neither R1 nor R2 ishydrogen if R4 andR3 together with the atoms to which they are attached forma4 , 5 or 6 membered heterocyclic ring. In this embodiment, there is provided a compound having a t of about1 hour in PBS under physiological conditions, wherein
R1和R2独立地为C1-C18烷基或芳基；或者R1和R2通过-(CH2)p-连接，其中p为2-9；R1 and R2 are independently C1 -C18 alkyl or aryl; or R1 and R2 are connected by -(CH2 )p -, wherein p is 2-9;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R4和R8各自为氢；和R and Rare each hydrogen; and
R5为胺。R5 is an amine.
在其它实施方案中，具有在N端连接的前药元件且t1/2为例如约1小时的前药包含具有以下结构的二肽前药元件：In other embodiments, a prodrug having an N-terminally linked prodrug element with a t of, for example, about1 hour comprises a dipeptide prodrug element having the structure:
其中in
R1和R2独立地为C1-C18烷基或(C0-C4烷基)(C6-C10芳基)R7；或者R1和R2通过-(CH2)p连接，其中p为2-9；R1 and R2 are independently C1 -C18 alkyl or (C0 -C4 alkyl) (C6 -C10 aryl) R7 ; or R1 and R2 pass through -(CH2 )p Connect, where p is 2-9;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R4和R8各自为氢；R and Rare each hydrogen;
R5为NH2；和R5 is NH2 ; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
或者，提供包含GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)的A链序列和X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)的B链序列的胰岛素前药，其中二肽前药元件与肽SEQ ID NO：3或SEQ ID NO：14的N端氨基酸的α氨基连接，且在生理条件下和PBS中显示介于约6-约24小时之间的t1/2。在一个实施方案中，这类化合物包含式I前药元件，其中Alternatively, an insulin prodrug comprising the A chain sequence of GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) and the B chain sequence of X14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14) is provided , wherein the dipeptide prodrug element is linked to the alpha amino group of the N-terminal amino acid of peptide SEQ ID NO: 3 or SEQ ID NO: 14, and exhibits a t between about 6 and about 24 hours under physiological conditions and in PBS1/2 . In one embodiment, such compounds comprise a prodrug element of formula I, wherein
R1和R2独立选自氢、C1-C18烷基和芳基，或者R1和R2通过-(CH2)p-连接，其中p为2-9；R1 and R2 are independently selected from hydrogen, C1 -C18 alkyl and aryl, or R1 and R2 are connected by -(CH2 )p -, wherein p is 2-9;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-12元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-12 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立选自氢、C1-C8烷基和芳基；和R and R are independently selected from hydrogen, C1-C 8alkyl and aryl; and
R5为胺，前提条件是R1和R2两者都不为氢，且前提条件是R4或R8之一为氢。R5 is an amine, with the proviso that neither R1 nor R2 ishydrogen , and with the proviso thatone ofR4 orR8 is hydrogen.
在另一个实施方案中，提供包含GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)的A链序列和X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)的B链序列的胰岛素前药，其中二肽前药元件与肽SEQ ID NO：3或SEQ ID NO：14的N端氨基酸的α氨基连接，且在生理条件下和PBS中显示约72-约168小时的t1/2。在一个实施方案中，这类化合物包含式I前药元件，其中In another embodiment, an A chain sequence comprising GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) and a B chain of X14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14) is provided The insulin prodrug of sequence, wherein dipeptide prodrug element and peptide SEQ ID NO:3 or the α amino group of the N-terminal amino acid of SEQ ID NO:14 are connected, and show about 72-about 168 hours of activity under physiological condition and PBS t1/2 . In one embodiment, such compounds comprise a prodrug element of formula I, wherein
R1选自氢、C1-C8烷基和芳基；R1 is selected from hydrogen, C1 -C8 alkyl and aryl;
R2为H；R2 is H;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R4和R8各自为氢；和R and Rare each hydrogen; and
R5为胺或N-取代的胺或羟基；R5 is amine or N-substituted amine or hydroxyl;
前提条件是，如果R1为烷基或芳基，则R1和R5与它们所连接的原子一起形成4-11元杂环。The proviso is that if R1 is alkyl or aryl, then R1 and R5together with the atoms to which they are attached forma 4-11 membered heterocyclic ring.
在一个实施方案中，具有与胰岛素A链或B链肽的N端α氨基酸连接的二肽前药元件且t1/2为例如介于约12-约72小时之间或者在一个实施方案中介于约12-约48小时之间的前药，包含具有以下结构的二肽前药元件：In one embodiment, there is a dipeptide prodrug element linked to the N-terminal alpha amino acid of the insulin A or B chain peptide and the t1/2 is, for example, between about 12 to about 72 hours or in one embodiment between Prodrugs between about 12 and about 48 hours, comprising a dipeptide prodrug element having the following structure:
其中R1和R2独立选自氢、C1-C18烷基、(C1-C18烷基)OH、(C1-C4烷基)NH2、和(C0-C4烷基)(C6-C10芳基)R7，或者R1和R2通过(CH2)p连接，其中p为2-9；wherein R1 and R2 are independently selected from hydrogen, C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C4 alkyl) NH2 , and (C0 -C4 alkane Base) (C6 -C10 aryl) R7 , or R1 and R2 are linked by (CH2 )p , where p is 2-9;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-12元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-12 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立选自氢、C1-C8烷基和(C0-C4烷基)(C6-C10芳基)R7；R4 and R8 are independently selected from hydrogen, C1 -C8 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R5为NH2；和R5 is NH2 ; and
R7选自H、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素；前提条件是R1和R2两者都不为氢，且前提条件是R4或R8中的至少一个为氢。R7 is selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen; with the proviso that neither R1 nor R2 ishydrogen , and with the proviso that at leastone ofR4 orR8 is hydrogen .
在一个实施方案中，具有与胰岛素A链或B链肽的N端氨基酸连接的二肽前药元件且t1/2为例如介于约12-约72小时之间，或者在一个实施方案中介于约12-约48小时之间的前药，包含具有以下结构的二肽前药元件：In one embodiment, there is a dipeptide prodrug element linked to the N-terminal amino acid of the insulin A or B chain peptide and the t1/2 is, for example, between about 12 to about 72 hours, or in one embodiment between Prodrugs between about 12 and about 48 hours, comprising a dipeptide prodrug element having the following structure:
其中R1和R2独立选自氢、C1-C8烷基和(C1-C4烷基)NH2，或者R1和R2通过(CH2)p连接，其中p为2-9；wherein R1 and R2 are independently selected from hydrogen, C1 -C8 alkyl and (C1 -C4 alkyl)NH2 , or R1 and R2 are linked by (CH2 )p , wherein p is 2- 9;
R3为C1-C8烷基，或者R3和R4与它们所连接的原子一起形成4-6元杂环；R3 is a C1 -C8 alkyl group, or R3 and R4 form a 4-6 membered heterocyclic ring together with the atoms they are connected to;
R4选自氢和C1-C8烷基；和R4 is selected from hydrogen and C1 -C8 alkyl; and
R5为NH2，前提条件是R1和R2两者都不为氢。R5 is NH2 with the proviso that neither R1 nor R2 is hydrogen.
在其它实施方案中，具有与胰岛素A链或B链肽的N端氨基酸连接的二肽前药元件且t1/2为例如介于约12-约72小时之间，或者在一个实施方案中介于约12-约48小时之间的前药，包含具有以下结构的二肽前药元件：In other embodiments, there is a dipeptide prodrug element linked to the N-terminal amino acid of the insulin A or B chain peptide and the t1/2 is, for example, between about 12 to about 72 hours, or in one embodiment between Prodrugs between about 12 and about 48 hours, comprising a dipeptide prodrug element having the following structure:
其中in
R1和R2独立选自氢、C1-C8烷基和(C1-C4烷基)NH2；R1 and R2 are independently selected from hydrogen, C1 -C8 alkyl and (C1 -C4 alkyl) NH2 ;
R3为C1-C6烷基；R3 is C1 -C6 alkyl;
R4为氢；和R4 is hydrogen; and
R5为NH2，前提条件是R1和R2两者都不为氢。R5 is NH2 with the proviso that neither R1 nor R2 is hydrogen.
在一个实施方案中，具有与胰岛素A链或B链肽的N端氨基酸连接的二肽前药元件且t1/2为例如介于约12-约72小时之间，或者在一个实施方案中介于约12-约48小时之间的前药，包含具有以下结构的二肽前药元件：In one embodiment, there is a dipeptide prodrug element linked to the N-terminal amino acid of the insulin A or B chain peptide and the t1/2 is, for example, between about 12 to about 72 hours, or in one embodiment between Prodrugs between about 12 and about 48 hours, comprising a dipeptide prodrug element having the following structure:
其中in
R1和R2独立选自氢和C1-C8烷基、(C1-C4烷基)NH2，或者R1和R2通过(CH2)p连接，其中p为2-9；R1 and R2 are independently selected from hydrogen and C1 -C8 alkyl, (C1 -C4 alkyl)NH2 , or R1 and R2 are linked by (CH2 )p , where p is 2-9 ;
R3为C1-C8烷基；R3 is C1 -C8 alkyl;
R4为(C0-C4烷基)(C6-C10芳基)R7；R4 is (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R5为NH2；和R5 is NH2 ; and
R7选自氢、C1-C8烷基和(C0-C4烷基)OH，前提条件是R1和R2两者都不为氢。R7 is selected from hydrogen, C1 -C8 alkyl and (C0 -C4 alkyl)OH, with the proviso that neither R1 nor R2 is hydrogen.
另外，提供具有与胰岛素A链或B链肽的N端α氨基酸连接的二肽前药元件且t1/2为例如约72-约168小时的前药，其中二肽前药元件具有以下结构：In addition, there is provided a prodrug having a dipeptide prodrug element linked to the N-terminal alpha amino acid of the insulin A-chain or B-chain peptide and having a t1/2 of, for example, about 72 to about 168 hours, wherein the dipeptide prodrug element has the structure :
其中R1选自氢、C1-C8烷基和(C0-C4烷基)(C6-C10芳基)R7；Wherein R1 is selected from hydrogen, C1 -C8 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R4和R8各自为氢；R and Rare each hydrogen;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素，前提条件是，如果R1为烷基或(C0-C4烷基)(C6-C10芳基)R7，则R1和R5与它们所连接的原子一起形成4-11元杂环。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl)NH2 , (C0 -C4 alkyl)OH and halogen, with the proviso that if R1 is alkyl or (C0 -C4 alkyl)(C6 -C10 aryl)R7 , then R1 and R5 form a 4-11 membered heterocyclic ring together with the atoms they are connected to.
在一个实施方案中，二肽前药元件与胰岛素肽内部氨基酸的侧链胺连接。在这个实施方案中，t1/2为例如约1小时的前药具有以下结构：In one embodiment, the dipeptide prodrug element is linked to the side chain amine of an internal amino acid of the insulin peptide. In this embodiment, the prodrug with a t of, for example, about1 hour has the following structure:
其中in
R1和R2独立地为C1-C8烷基或(C0-C4烷基)(C6-C10芳基)R7；或者R1和R2通过-(CH2)p-连接，其中p为2-9；R1 and R2 are independently C1 -C8 alkyl or (C0 -C4 alkyl) (C6 -C10 aryl) R7 ; or R1 and R2 pass through -(CH2 )p - connection, where p is 2-9;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R4和R8各自为氢；R and Rare each hydrogen;
R5为NH2；和R5 is NH2 ; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
此外，t1/2为例如介于约6-约24小时之间且具有与内部氨基酸侧链连接的二肽前药元件的前药包含具有以下结构的二肽前药元件：In addition, prodrugs having a t1/2 of, for example, between about 6 to about 24 hours and having a dipeptide prodrug element attached to an internal amino acid side chain comprise a dipeptide prodrug element having the following structure:
其中R1和R2独立选自氢、C1-C8烷基和(C0-C4烷基)(C6-C10芳基)R7，或者R1和R2通过-(CH2)p-连接，其中p为2-9；wherein R1 and R2 are independently selected from hydrogen, C1 -C8 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 , or R1 and R2 are passed through -(CH2 )p -connection, where p is 2-9;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-12元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-12 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立地为氢、C1-C18烷基或(C0-C4烷基)(C6-C10芳基)R7；R4 and R8 are independently hydrogen, C1 -C18 alkyl or (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R5为NHR6；R5 is NHR6 ;
R6为H或C1-C8烷基，或者R6和R2与它们所连接的原子一起形成4、5或6元杂环；和R6 is H or C1 -C8 alkyl, or R6 and R2 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素，前提条件是R1和R2两者都不为氢，且前提条件是R4或R8中的至少一个为氢。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl)NH2 , (C0 -C4 alkyl)OH and halogen, with the proviso that neither R nor R ishydrogen , and with theproviso that at leastone ofR or R is hydrogen .
另外，提供t1/2为例如约72-约168小时且具有与胰岛素肽的内部氨基酸侧链连接的二肽前药元件的前药，其中所述二肽前药元件具有以下结构：Additionally,prodrugs having a t of, for example, about 72 to about 168 hours and having a dipeptide prodrug element linked to an internal amino acid side chain of the insulin peptide are provided, wherein the dipeptide prodrug element has the structure:
其中R1选自氢、C1-C18烷基和(C0-C4烷基)(C6-C10芳基)R7；Wherein R1 is selected from hydrogen, C1 -C18 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R3为C1-C18烷基；R3 is C1 -C18 alkyl;
R4和R8各自为氢；R and Rare each hydrogen;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H或C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H or C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素；前提条件是，如果R1和R2两个均独立地为烷基或(C0-C4烷基)(C6-C10芳基)R7，则R1或R2中的任一个通过(CH2)p与R5连接，其中p为2-9。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl) NH2 , (C0 -C4 alkyl) OH and halogen; with the proviso that if R1 and R2 are both independently alkyl or (C0 -C4 alkyl)(C6 -C10 aryl) R7 , then any one of R1 or R2 is connected to R5 through (CH2 )p , wherein p is 2-9.
在一个实施方案中，二肽前药元件与胰岛素肽内部氨基酸的侧链胺连接，其中内部氨基酸包含下式IV的结构：In one embodiment, the dipeptide prodrug element is linked to the side chain amine of an internal amino acid of the insulin peptide, wherein the internal amino acid comprises the structure of Formula IV below:
其中in
n为选自1-4的整数。在一个实施方案中，n为3或4，而在一个实施方案中，内部氨基酸为赖氨酸。在一个实施方案中，二肽前药元件与位于胰岛素肽B链28或29位的氨基酸侧链上的伯胺连接。n is an integer selected from 1-4. In one embodiment, n is 3 or 4, and in one embodiment, the internal amino acid is lysine. In one embodiment, the dipeptide prodrug element is linked to a primary amine on the side chain of the amino acid at position 28 or 29 of the insulin peptide B chain.
在一个实施方案中，提供包含A链序列和B链序列的胰岛素前药，其中A链包含序列GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)，B链序列包含序列X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)，其中In one embodiment, there is provided an insulin prodrug comprising an A chain sequence comprising the sequence GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) and a B chain sequence comprising the sequence X14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14), wherein
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NH2、NHR10和OCH3，其中R10为H或包含以下通用结构的二肽：wherein X is selected from OH, NH2 , NHR10 and OCH3 , wherein R10 is H or a dipeptide comprising the following general structure:
其中in
R1和R2独立选自氢、C1-C8烷基和(C0-C4烷基)(C6-C10芳基)R7，或者R1和R2通过-(CH2)p-连接，其中p为2-9；R1 and R2 are independently selected from hydrogen, C1 -C8 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 , or R1 and R2 are passed through -(CH2 )p - connection, where p is 2-9;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-12元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-12 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立地为氢、C1-C18烷基或(C0-C4烷基)(C6-C10芳基)R7；R4 and R8 are independently hydrogen, C1 -C18 alkyl or (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R5为NHR6；R5 is NHR6 ;
R6为H或C1-C8烷基，或者R6和R2与它们所连接的原子一起形成4、5或6元杂环；和R6 is H or C1 -C8 alkyl, or R6 and R2 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素；R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl)NH2 , (C0 -C4 alkyl)OH and halogen;
X3选自天冬酰胺、甘氨酸、丙氨酸、苏氨酸和丝氨酸；X is selected fromasparagine , glycine, alanine, threonine and serine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X7为以下通用结构的氨基酸X7 is an amino acid of the following general structure
其中X12选自OH、NH2、NHR11和OCH3，其中R11为H或包含以下通用结构的二肽：Wherein X12 is selected from OH, NH2 , NHR11 and OCH3 , wherein R11 is H or a dipeptide comprising the following general structure:
X8为以下通用结构的氨基酸X8 is an amino acid of the following general structure
其中X13选自H、OH、NH2、NHR12和OCH3，其中R12为H或包含以下通用结构的二肽：Wherein X13 is selected from H, OH, NH2 , NHR12 and OCH3 , wherein R12 is H or a dipeptide comprising the following general structure:
X14选自氢(形成N端胺)、X9VNQ(SEQ ID NO：21)、VNQ、NQ和Q，X14 is selected from hydrogen (forming N-terminal amine), X9 VNQ (SEQ ID NO: 21), VNQ, NQ and Q,
X9选自苯丙氨酸和脱氨基-苯丙氨酸，前提条件是R10、R11和R12中的一个并且只有一个为包含以下通用结构的二肽：X9 is selected from phenylalanine and desamino-phenylalanine with the proviso that one and onlyone of R10,R11 andR12 is a dipeptide comprising the following general structure:
根据其中二肽前药元件与芳族氨基酸芳基的氨基取代基连接的一个实施方案，可提供具有所需激活时间的前药制剂。例如，提供包含下式III结构且在生理条件下和PBS中t1/2约为1小时的胰岛素前药：According to one embodiment wherein the dipeptide prodrug element is attached to the amino substituent of the aryl group of the aromatic amino acid, a prodrug formulation with a desired activation time can be provided. For example, insulin prodrugs comprising the structure of formula III and having a t1/2 of about 1 hour under physiological conditions and in PBS are provided:
其中m为0-3的整数。在其中胰岛素前药包含式III结构并具有这类半寿期的一个实施方案中，Wherein m is an integer of 0-3. In one embodiment wherein the insulin prodrug comprises the structure of formula III and has such a half-life,
R1和R2独立地为C1-C18烷基或芳基；R1 and R2 are independently C1 -C18 alkyl or aryl;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-12元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-12 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立选自氢、C1-C18烷基和芳基；和R and R are independently selected from hydrogen, C1-C 18alkyl and aryl; and
R5为胺或羟基。在一个实施方案中，m为1。R5 is amine or hydroxyl. In one embodiment, m is 1.
在一个实施方案中，二肽前药元件通过胰岛素肽芳族氨基酸芳基上存在的胺与胰岛素肽连接，其中t1/2为例如约1小时的前药具有以下结构的二肽：In one embodiment, the dipeptide prodrug element is linked to the insulin peptide through an amine present on the aryl group of the aromatic amino acid of the insulin peptide, wherein t1/2 is, for example, about 1 hour. The prodrug has a dipeptide of the following structure:
其中R1和R2独立地为C1-C18烷基或(C0-C4烷基)(C6-C10芳基)R7；Wherein R1 and R2 are independently C1 -C18 alkyl or (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-12元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-12 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立选自氢、C1-C18烷基和(C0-C4烷基)(C6-C10芳基)R7；R4 and R8 are independently selected from hydrogen, C1 -C18 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R5为NH2或OH；和R5 is NH2 or OH; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
在另一个实施方案中，提供包含式III结构且在生理条件下和PBS中t1/2为约6-约24小时的胰岛素前药，其中m为0-3的整数。在其中胰岛素前药包含式III结构并具有这类半寿期的一个实施方案中，In another embodiment, there is provided an insulin prodrug comprising the structure of formula III and having a t1/2 of about 6 to about 24 hours under physiological conditions and in PBS, wherein m is an integer of 0-3. In one embodiment wherein the insulin prodrug comprises the structure of formula III and has such a half-life,
R1和R2独立选自氢、C1-C18烷基和芳基，或者R1和R2通过-(CH2)p-连接，其中p为2-9；R1 and R2 are independently selected from hydrogen, C1 -C18 alkyl and aryl, or R1 and R2 are connected by -(CH2 )p -, wherein p is 2-9;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-6元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-6 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立选自氢、C1-C18烷基和芳基；和R and R are independently selected from hydrogen, C1-C 18alkyl and aryl; and
R5为胺或N-取代的胺。在一个实施方案中，m为1。R5 is an amine or an N-substituted amine. In one embodiment, m is 1.
在一个实施方案中，提供具有通过存在于芳族氨基酸芳基上的胺连接的二肽前药元件且t1/2为例如约6-约24小时的前药，其中所述二肽包含以下结构：In one embodiment, there is provided a prodrug having a dipeptide prodrug element linked through an amine present on the aryl of an aromatic amino acid and a t1/2 of, for example, from about 6 to about 24 hours, wherein the dipeptide comprises structure:
其中in
R1选自氢、C1-C18烷基、(C1-C18烷基)OH、(C1-C4烷基)NH2和(C0-C4烷基)(C6-C10芳基)R7；R1 is selected from hydrogen, C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C4 alkyl) NH2 and (C0 -C4 alkyl) (C6 - C10 aryl) R7 ;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-6元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-6 membered heterocyclic ring together with the atoms they are connected to;
R4和R8独立选自氢、C1-C18烷基和(C0-C4烷基)(C6-C10芳基)R7；R4 and R8 are independently selected from hydrogen, C1 -C18 alkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 ;
R5为NHR6；R5 is NHR6 ;
R6为H、C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
在另一个实施方案中，提供包含式III结构且在生理条件下和PBS中t1/2为约72-约168小时的胰岛素前药，其中m为0-3的整数。在其中胰岛素前药包含式III结构并具有这类半寿期的一个实施方案中，In another embodiment, there is provided an insulin prodrug comprising the structure of formula III and having a t1/2 of about 72 to about 168 hours under physiological conditions and in PBS, wherein m is an integer from 0-3. In one embodiment wherein the insulin prodrug comprises the structure of formula III and has such a half-life,
R1和R2独立选自氢、C1-C8烷基和芳基；R1 and R2 are independently selected from hydrogen, C1 -C8 alkyl and aryl;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-6杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-6 heterocyclic ring together with the atoms they are connected to;
R4和R8各自为氢；和R and Rare each hydrogen; and
R5选自胺、N-取代的胺和羟基。在一个实施方案中，m为1。R5 is selected from amines, N-substituted amines and hydroxyl groups. In one embodiment, m is 1.
在一个实施方案中，提供具有通过芳族氨基酸连接的二肽前药元件且t1/2为例如约72-约168小时的前药，其中所述二肽包含以下结构：In one embodiment, there is provided a prodrug having a dipeptide prodrug element linked by an aromatic amino acid and at of, for example, about 72 to about 168 hours, wherein the dipeptide comprises the structure:
其中R1选自氢、C1-C8烷基、(C1-C4烷基)COOH和(C0-C4烷基)(C6-C10芳基)R7，或者R1和R5与它们所连接的原子一起形成4-11元杂环；wherein R1 is selected from hydrogen, C1 -C8 alkyl, (C1 -C4 alkyl) COOH and (C0 -C4 alkyl) (C6 -C10 aryl) R7 , or R1 and Rtogether with the atoms to which they are attached form a 4-11 membered heterocyclic ring;
R3为C1-C18烷基，或者R3和R4与它们所连接的原子一起形成4-6元杂环；R3 is a C1 -C18 alkyl group, or R3 and R4 form a 4-6 membered heterocyclic ring together with the atoms they are connected to;
R4为氢或与R3形成4-6元杂环；R4 is hydrogen or forms a 4-6 membered heterocycle with R3 ;
R8为氢；R is hydrogen;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H或C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H or C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
在一个实施方案中，胰岛素前药类似物包含GIVEQCCX1SICSLYQLENX2CX3-R13(SEQID NO：3)的A链序列和X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)的B链序列，其中In one embodiment, the insulin prodrug analog comprises the A chain sequence of GIVEQCCX1 SICSLYQLENX2 CX3 -R13 (SEQ ID NO: 3) and X14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14) the B chain sequence, wherein
X1选自苏氨酸、组氨酸、精氨酸和赖氨酸；X is selected from threonine, histidine, arginine and lysine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NH2和OCH3；Wherein X is selected from OH, NH2 and OCH3 ;
X3为天冬酰胺、甘氨酸、丙氨酸、苏氨酸或丝氨酸。X3 is asparagine, glycine, alanine, threonine or serine.
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X7为酪氨酸；X7 is tyrosine;
X8为酪氨酸或苯丙氨酸；X8 is tyrosine or phenylalanine;
X9选自苯丙氨酸和脱氨基-苯丙氨酸；X is selected from phenylalanine anddesamino -phenylalanine;
X10为天冬氨酸-赖氨酸二肽、赖氨酸-脯氨酸二肽或脯氨酸-赖氨酸二肽；X10 is aspartic acid-lysine dipeptide, lysine-proline dipeptide or proline-lysine dipeptide;
X11为苏氨酸、丙氨酸或苏氨酸-精氨酸-精氨酸三肽；而且其中B链包含1-4个氨基酸的羧基端延伸物，其中所述羧基端延伸物包含具有以下结构的氨基酸：X isthreonine , alanine or threonine-arginine-arginine tripeptide; and wherein the B chain comprises a carboxy-terminal extension of 1-4 amino acids, wherein the carboxy-terminal extension comprises a Amino acids with the following structure:
其中m为0-3的整数；Where m is an integer of 0-3;
n为1-4的整数；n is an integer of 1-4;
R12为包含以下通用结构的二肽：R12 is a dipeptide comprising the following general structure:
R1选自H和C1-C8烷基；和R1 is selected from H and C1 -C8 alkyl; and
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 and CH2 (C5 -C9 heteroaryl);
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；R6 is H, or R6 and R2 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素；和R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C4 alkyl)NH2 , (C0 -C4 alkyl)OH and halogen; and
R13为COOH或CONH2。R13 is COOH or CONH2 .
在一个实施方案中，胰岛素前药类似物包含A链序列和B链序列，所述A链序列包括序列GIVEQCCX1SICSLYQLENX2CX3-R13(SEQ ID NO：3)，所述B 链序列包括序列J-X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)或J-X9VNQX4LCGX5X6LVEALX7LVCGERGFX8YTX10X11-R14(SEQ ID NO：5)，其中J为H(形成N端胺)或包含以下通用结构的二肽：In one embodiment, the insulin prodrug analog comprises an A chain sequence comprising the sequence GIVEQCCX1 SICSLYQLENX2 CX3 -R13 (SEQ ID NO: 3), and a B chain sequence comprising The sequence JX14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14) or JX9 VNQX4 LCGX5 X6 LVEALX7 LVCGERGFX8 YTX10 X11 -R14 (SEQ ID NO: 5), wherein J is H (forming an N-terminal amine) or a dipeptide comprising the following general structure:
其中X14或是将“J”元件与SEQ ID NO：14连接的键，或者X14表示选自以下的将“J”元件与SEQ ID NO：14连接的1-4个氨基酸的序列：FVNQ(SEQ ID NO：11)、VNQ、NQ和Q；wherein X14 is either a bond linking the "J" element to SEQ ID NO: 14, or X14 represents a sequence of 1-4 amino acids selected from the group consisting of the following linking the "J" element to SEQ ID NO: 14: FVNQ (SEQ ID NO: 11), VNQ, NQ and Q;
X1选自苏氨酸、组氨酸、精氨酸和赖氨酸；X is selected from threonine, histidine, arginine and lysine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NH2和OCH3；Wherein X is selected from OH, NH2 and OCH3 ;
X3为天冬酰胺、甘氨酸、丙氨酸、苏氨酸或丝氨酸。X3 is asparagine, glycine, alanine, threonine or serine.
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X7为以下通用结构的氨基酸X7 is an amino acid of the following general structure
其中X12选自OH、OCH3、NH2和NHR11，其中R11为包含以下通用结构的二肽：Wherein X12 is selected from OH, OCH3 , NH2 and NHR11 , wherein R11 is a dipeptide comprising the following general structure:
X8为以下通用结构的氨基酸X8 is an amino acid of the following general structure
其中X13选自H、OH、OCH3、NH2和NHR12，其中R12为包含以下通用结构的二肽：Wherein X13 is selected from H, OH, OCH3 , NH2 and NHR12 , wherein R12 is a dipeptide comprising the following general structure:
X9选自苯丙氨酸和脱氨基-苯丙氨酸；X is selected from phenylalanine anddesamino -phenylalanine;
X10为天冬氨酸-赖氨酸二肽、赖氨酸-脯氨酸二肽或脯氨酸-赖氨酸二肽；X10 is aspartic acid-lysine dipeptide, lysine-proline dipeptide or proline-lysine dipeptide;
X11为苏氨酸、丙氨酸或苏氨酸-精氨酸-精氨酸三肽；X11 is threonine, alanine or threonine-arginine-arginine tripeptide;
其中R1选自H和C1-C8烷基；和wherein R is selected from H and C1-C 8alkyl ; and
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 and CH2 (C5 -C9 heteroaryl);
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；R6 is H, or R6 and R2 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R7选自H和OH；和Ris selected from H and OH; and
R13和R14独立地为COOH或CONH2，前提条件是X12、X13或J中的一个并且只有一个为包含以下通用结构的二肽：R13 and R14 are independently COOH or CONH2 , provided that one and only one of X12 , X13 or J is a dipeptide comprising the following general structure:
(即只有一个二肽前药元件与胰岛素肽连接)。在一个实施方案中，J为包含以下通用结构的二肽： (ie only one dipeptide prodrug element is linked to the insulin peptide). In one embodiment, J is a dipeptide comprising the general structure:
X和X12各自为OH，X13为H，进一步的前提条件是如果R4和R3与它们所连接的原子一起形成5元杂环，则R1和R2两者都不为H。在一个备选实施方案中，X12为NHR11，J和X13各自为H，X为OH。在另一个备选实施方案中，X13为NHR12，X和X12各自为OH，J为H。在一个实施方案中，B链包含序列J-X9VNQX4LCGX5X6LVEALX7LVCGERGFX8YTPKT(SEQ ID NO：15)或J-X9VNQX4LCGX5X6LVEALX7LVCGERGFX8YTKPT(SEQ ID NO：16)，其中J、X4、X5、X6、X7、X8和X9如紧接的上文中所定义。在另一个实施方案中，R3为C1-C6烷基，R4选自H、C1-C4烷基，或者R3和R4与它们所连接的原子一起形成5元杂环。在另一个实施方案中，X4为组氨酸，X5为丝氨酸，X6为组氨酸。 X andX12 are each OH andX13 is H, with the further proviso that neither R1 nor R2 is H if R4 andR3 together with the atoms to which they are attached forma5- membered heterocyclic ring. In an alternative embodiment,X12 isNHR11 , J andX13 are each H, and X is OH. In another alternative embodiment,X13 isNHR12 , X andX12 are each OH, and J is H. In one embodiment, the B chain comprises the sequence JX9 VNQX4 LCGX5 X6 LVEALX7 LVCGERGFX8 YTPKT (SEQ ID NO: 15) or JX9 VNQX4 LCGX5 X6 LVEALX7 LVCGERGFX8 YTKPT (SEQ ID NO: 16 ), wherein J, X4 , X5 , X6 , X7 , X8 and X9 are as defined immediately above. In another embodiment, R3 is C1 -C6 alkyl, R4 is selected from H, C1 -C4 alkyl, or R3 and R4 together with the atoms to which they are attached form a 5 membered heterocyclic ring .In another embodiment,X4 is histidine, X5 is serine, andX6 is histidine.
在另一个实施方案中，提供包含Z-GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)的A链序列和包含序列X4LCGX5X6LVEALYLVCGERGFF(SEQ ID NO：4)的B链序列的胰岛素前药类似物，其中In another embodiment, there is provided an A-chain sequence comprising Z-GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) and a B-chain sequence comprising the sequence X4 LCGX5 X6 LVEALYLVCGERGFF (SEQ ID NO: 4). Insulin prodrug analogues, of which
Z为H或包含以下通用结构的二肽：Z is H or a dipeptide comprising the following general structure:
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NH2、NHR10和OCH3，其中R10为包含以下通用结构的二肽：wherein X is selected from OH, NH2 , NHR10 and OCH3 , wherein R10 is a dipeptide comprising the following general structure:
X3选自天冬酰胺、甘氨酸、丙氨酸、苏氨酸或丝氨酸；X is selected fromasparagine , glycine, alanine, threonine or serine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
其中R1选自H和C1-C8烷基；Wherein R1 is selected from H and C1 -C8 alkyl;
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7、CH2(C5-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C6环烷基；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 , CH2 (C5 -C9 heteroaryl), or R1 and R2 together with the atoms they are connected to form a C3 -C6 cycloalkyl group;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C3 -C6 ) cycloalkyl, or R4 and R3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclic ring;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, or R6 and R2together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring; and
R7选自H和OH，前提条件是X和Z不同时为二肽且X为OH时Z不为H。在一个实施方案中，如果Z为包含以下通用结构的二肽：R7 is selected from H and OH with the proviso that X and Z are not both dipeptides and Z is not H when X is OH. In one embodiment, if Z is a dipeptide comprising the general structure:
且R4和R3与它们所连接的原子一起形成5元杂环，则R1和R2中的至少一个不为H。在一个实施方案中，A链包含序列Z-GIVEQCCX1SICSLYQLENYCX3(SEQ IDNO：17)，B链序列包含序列X9VNQX4LCGX5X6LVEALYLVCGERGFFYTPKT(SEQ ID NO：12)或X9VNQX4LCGX5X6LVEALYLVCGERGFFYTKPT(SEQ ID NO：13)。 and R4 and R3 together with the atoms to which they are attached form a 5-membered heterocyclic ring, then at least one of R1 and R2 is not H. In one embodiment, the A chain comprises the sequence Z-GIVEQCCX1 SICSLYQLENYCX3 (SEQ ID NO: 17), and the B chain sequence comprises the sequence X9 VNQX4 LCGX5 X6 LVEALYLVCGERGFFYTPKT (SEQ ID NO: 12) or X9 VNQX4 LCGX5 X6 LVEALYLVCGERGFFYTKPT (SEQ ID NO: 13).
在一个备选实施方案中，提供包含GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)的A链序列和含有序列X9VNQX4LCGX5X6LVEALYLVCGERGFFYTPKT(SEQ ID NO：12)或X9VNQX4LCGX5X6LVEALYLVCGERGFFYTKPT(SEQ ID NO：13)的B链序列的胰岛素前药类似物，其中X1选自苏氨酸和组氨酸；In an alternative embodiment, there is provided an A chain sequence comprising GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) and a sequence comprising the sequence X9 VNQX4 LCGX5 X6 LVEALYLVCGERGFFYTPKT (SEQ ID NO: 12) or X9 VNQX4 LCGX5 X6 LVEALYLVCGERGFFYTKPT (SEQ ID NO: 13) B chain sequence insulin prodrug analogs, wherein X1 is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中U为氨基酸或羟酸，O为N-烷基化氨基酸；Wherein U is an amino acid or a hydroxy acid, and O is an N-alkylated amino acid;
X3为天冬酰胺、甘氨酸、丙氨酸、苏氨酸或丝氨酸；X3 is asparagine, glycine, alanine, threonine or serine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X9选自苯丙氨酸和脱氨基-苯丙氨酸。在一个实施方案中，U-O表示以下通用结构的二肽：X9 is selected from phenylalanine and desamino-phenylalanine. In one embodiment, UO represents a dipeptide of the following general structure:
其中R1选自H和C1-C8烷基；Wherein R1 is selected from H and C1 -C8 alkyl;
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7、CH2(C5-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C6环烷基；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 , CH2 (C5 -C9 heteroaryl), or R1 and R2 together with the atoms they are connected to form a C3 -C6 cycloalkyl group;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, or R6 and R2together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring; and
R7选自H和OH。在另一个实施方案中，X7为酪氨酸，X8为苯丙氨酸，X9为苯丙氨酸，而在又一个实施方案中，X4为组氨酸，X5为丝氨酸，X6为组氨酸。在另一个实施方案中，U-O表示以下通用结构的二肽：R7 is selected from H and OH.In another embodiment, X7 is tyrosine,X8 is phenylalanine,X9 is phenylalanine, and in yet another embodiment,X4 is histidine,X5 is serine, X6 is histidine. In another embodiment, UO represents a dipeptide of the following general structure:
其中in
R1、R2、R4和R8独立选自H、C1-C18烷基、C2-C18烯基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7、(C1-C4烷基)(C3-C9杂芳基)和C1-C12烷基(W1)C1-C12烷基，其中W1为选自N、S和O的杂原子，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基；或者R4和R8与它们所连接的原子一起形成C3-C6环烷基；R1 , R2 , R4 and R8 are independently selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C1 -C18 alkyl)OH, (C1 -C18 alkane base) SH, (C2 -C3 alkyl) SCH3 , (C1 -C4 alkyl) CONH2 , (C1 -C4 alkyl) COOH, (C1 -C4 alkyl) NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 , (C1 -C4 alkyl) (C3 -C9 heteroaryl) and C1 -C12 alkyl (W1 )C1 -C12 alkyl, wherein W1 is a heteroatom selected from N, S and O, or R1 and R2 together with the atoms to which they are attached form C3 -C12 cycloalkyl; or R4 and R8 form C3 -C6 cycloalkyl together with the atoms they are connected to;
R3选自C1-C18烷基、(C1-C18烷基)OH、(C1-C18烷基)NH2、(C1-C18烷基)SH、(C0-C4烷基)(C3-C6)环烷基、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和(C1-C4烷基)(C3-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成4、5或6元杂环；R3 is selected from C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C18 alkyl) NH2 , (C1 -C18 alkyl) SH, (C0 - C4 alkyl) (C3 -C6 ) cycloalkyl, (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and (C1 -C4 alkyl) (C3 -C9 heteroaryl), or R4 and R3 form a 4, 5 or 6 membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen.
在另一个实施方案中，提供包含Z-GIVEQCCTSICSLYQLENX2CX3-R13(SEQ ID NO：18)的A链序列和含有序列X4LCGSHLVEALYLVCGERGFF-R14(SEQ ID NO：19)的B链序列的胰岛素前药类似物，其中In another embodiment, an A-chain sequence comprising Z-GIVEQCCTSICSLYQLENX2 CX3 -R13 (SEQ ID NO: 18) and a B-chain sequence comprising the sequence X4 LCGSHLVEALYLVCGERGFF-R14 (SEQ ID NO: 19) are provided. Insulin prodrug analogues, of which
Z为H或包含以下通用结构的连接酰胺的二肽：Z is H or an amide-linked dipeptide comprising the general structure:
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH和NHR10；Wherein X is selected from OH and NHR10 ;
其中R10为包含以下通用结构的二肽：wherein R10 is a dipeptide comprising the following general structure:
X3为丝氨酸、天冬酰胺或甘氨酸；X3 is serine, asparagine or glycine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
R1选自H和C1-C8烷基；R1 is selected from H and C1 -C8 alkyl;
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7、CH2(C5-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C6环烷基；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 , CH2 (C5 -C9 heteroaryl), or R1 and R2 together with the atoms they are connected to form a C3 -C6 cycloalkyl group;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C3 -C6 ) cycloalkyl, or R4 and R3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclic ring;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；R6 is H, or R6 and R2 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R7选自H和OH；和Ris selected from H and OH; and
R13和R14独立地为COOH或CONH2；前提条件是如果Z为H，则X不为OH，且如果X为OH，则Z不为H。在一个实施方案中，R13为COOH，R14为CONH2。在另一个实施方案中，X2为下式III通用结构的氨基酸：R13 andR14 are independently COOH or CONH2; with the proviso that if Z is H, then X is not OH, and if X is OH, then Z is not H. In one embodiment,R13 is COOH andR14 isCONH2 . In another embodiment, X is an aminoacid of the general structure of Formula III below:
Z为H，X3为丝氨酸，X4为组氨酸，R13为COOH，R14为CONH2。在又一个实施方案中，Z is H, X3 is serine, X4 is histidine, R13 is COOH, R14 is CONH2 . In yet another embodiment,
R1选自H和C1-C4烷基；R1 is selected from H and C1 -C4 alkyl;
R2选自H、C1-C6烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)，或者R2和R6与它们所连接的原子一起形成5元杂环；R2 is selected from H, C1 -C6 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and CH2 (C5 -C9 heteroaryl), or R and R together with the atoms to which they are attached form a5- membered heterocyclic ring;
R3为C1-C6烷基；R3 is C1 -C6 alkyl;
R4选自H和C1-C4烷基，或者R3和R4与它们所连接的原子一起形成5元杂环。在另一个实施方案中，R3为CH3，R4为H，R5为NH2，或者，R5为NH2，R3和R4与它们所连接的原子一起形成5元杂环。根据一个实施方案，胰岛素前药类似物的B链包含序列FVNQHLCGSHLVEALYLVCGERGFFYTPKT-R14(SEQ ID NO：8)、FVNQHLCGSHLVEALYLVCGERGFFYTKPT-R14(SEQ ID NO：9)或FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR-R14(SEQ ID NO：10)，其中R14为COOH或CONH2，而在一个实施方案中，R14为CONH2。R4 is selected from H and C1 -C4 alkyl, or R3 and R4 together with the atoms to which they are attached form a 5-membered heterocyclic ring.In another embodiment,R3 isCH3 , R4 is H, R5 isNH2 , alternatively,R5 isNH2 , andR3 and R4 together with the atoms to which they are attached form a5membered heterocyclic ring. According to one embodiment, the B chain of the insulin prodrug analogue comprises the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT-R14 (SEQ ID NO: 8), FVNQHLCGSHLVEALYLVCGERGFFYTKPT-R14 (SEQ ID NO: 9) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR-R14 (SEQ ID NO: 10) , wherein R14 is COOH or CONH2 , and in one embodiment, R14 is CONH2 .
在一个实施方案中，提供包含序列Z-GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)的多肽的胰岛素前药类似物，其中In one embodiment, there is provided an insulin prodrug analogue comprising a polypeptide of the sequence Z-GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3), wherein
Z为包含以下通用结构的二肽：Z is a dipeptide comprising the following general structure:
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NH2和OCH3；Wherein X is selected from OH, NH2 and OCH3 ;
X3为天冬酰胺、甘氨酸、丙氨酸、苏氨酸或丝氨酸；X3 is asparagine, glycine, alanine, threonine or serine;
R1选自H和C1-C8烷基；和R1 is selected from H and C1 -C8 alkyl; and
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7、CH2(C5-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C6环烷基；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 , CH2 (C5 -C9 heteroaryl), or R1 and R2 together with the atoms they are connected to form a C3 -C6 cycloalkyl group;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, or R6 and R2together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring; and
R7选自H和OH。在一个实施方案中，X1为苏氨酸，X3为天冬酰胺或甘氨酸，而在另一个实施方案中，R3为C1-C6烷基，R4选自H、C1-C4烷基、(C3-C6)环烷基、(C1-C4烷基)OH、(C1-C4烷基)SH和(C0-C4烷基)(C6芳基)R7，或者R3和R4与它们所连接的原子一起形成5元杂环，前提条件是如果R4和R3与它们所连接的原子一起形成5或6元杂环，则R1和R2两个都不为H。R7 is selected from H and OH. In one embodiment, X1 is threonine, X3 is asparagine or glycine, and in another embodiment, R3 is C1 -C6 alkyl, R4 is selected from H, C1 - C4 alkyl, (C3 -C6 ) cycloalkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH and (C0 -C4 alkyl) (C6 aryl) R7 , or R3 and R4 together with the atoms to which they are attached form a 5-membered heterocyclic ring, provided that if R4 and R3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclic ring, thenNeitherR1 nor R2 is H.
在一个实施方案中，提供包含序列GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)的多肽的胰岛素前药类似物，其中In one embodiment, there is provided an insulin prodrug analogue comprising a polypeptide of the sequence GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3), wherein
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
X3为天冬酰胺、甘氨酸、丙氨酸、苏氨酸或丝氨酸；X3 is asparagine, glycine, alanine, threonine or serine;
R1选自H和C1-C8烷基；和R1 is selected from H and C1 -C8 alkyl; and
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7、CH2(C5-C9杂芳基)，或者R1和R2与它们所连接的原子一起形成C3-C6环烷基；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 , CH2 (C5 -C9 heteroaryl), or R1 and R2 together with the atoms they are connected to form a C3 -C6 cycloalkyl group;
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C3 -C6 ) cycloalkyl, or R4 and R3 together with the atoms to which they are attached form a 5- or 6-membered heterocyclic ring;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, or R6 and R2together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring; and
R7选自H和OH。在一个实施方案中，如紧接的上文中所定义的A链GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)通过二硫键或作为单链多肽与包含序列X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)的B链连接，其中R7 is selected from H and OH. In one embodiment, the A chain GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) as defined immediately above is combined with the sequence X14 -X4 LCGX5 X via a disulfide bond or as a single-chain polypeptide6 LVEALX7 LVCGERGFX8 (SEQ ID NO: 14) B chain connection, wherein
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X7为酪氨酸；和X7 is tyrosine; and
X8为苯丙氨酸。X8 is phenylalanine.
在另一个实施方案中，二肽前药元件具有式I的结构，其中In another embodiment, the dipeptide prodrug element has the structure of Formula I, wherein
R1、R2和R4独立选自H、C1-C18烷基、C2-C18烯基、(C1-C18烷基)OH、(C1-C18烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7、(C1-C4烷基)(C3-C9杂芳基)和C1-C12烷基(W1)C1-C12烷基，其中W1为选自N、S和O的杂原子，或者R1和R2与它们所连接的原子一起形成C3-C12环烷基；R1 , R2 and R4 are independently selected from H, C1 -C18 alkyl, C2 -C18 alkenyl, (C1 -C18 alkyl)OH, (C1 -C18 alkyl)SH , (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 , (C1 -C4 alkyl) (C3 -C9 heteroaryl) and C1 - C12 alkyl (W1 ) C1 -C12 alkyl, wherein W1 is a heteroatom selected from N, S and O, or R1 and R2 together with the atoms to which they are attached form C3 -C12 Cycloalkyl;
R3选自C1-C18烷基、(C1-C18烷基)OH、(C1-C18烷基)NH2、(C1-C18烷基)SH、(C0-C4烷基)(C3-C6)环烷基、(C0-C4烷基)(C2-C5杂环基)、(C0-C4烷基)(C6-C10芳基)R7和(C1-C4烷基)(C3-C9杂芳基)，或者R4和R3与它们所连接的原子一起形成4、5或6元杂环；R3 is selected from C1 -C18 alkyl, (C1 -C18 alkyl) OH, (C1 -C18 alkyl) NH2 , (C1 -C18 alkyl) SH, (C0 - C4 alkyl) (C3 -C6 ) cycloalkyl, (C0 -C4 alkyl) (C2 -C5 heterocyclyl), (C0 -C4 alkyl) (C6 -C10 aryl) R7 and (C1 -C4 alkyl) (C3 -C9 heteroaryl), or R4 and R3 form a 4, 5 or 6 membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H、C1-C8烷基，或者R6和R1与它们所连接的原子一起形成4、5或6元杂环；和R6 is H, C1 -C8 alkyl, or R6 and R1 together with the atoms to which they are attached form a 4, 5 or 6 membered heterocyclic ring; and
R7选自氢、C1-C18烷基、C2-C18烯基、(C0-C4烷基)CONH2、(C0-C4烷基)COOH、(C0-C4烷基)NH2、(C0-C4烷基)OH和卤素。在另一个实施方案中，R3为C1-C6烷基，R4选自H和C1-C4烷基，或者R3和R4与它们所连接的原子一起形成5元杂环。在一个实施方案中，X1为苏氨酸，X3为天冬酰胺或甘氨酸。R7 is selected from hydrogen, C1 -C18 alkyl, C2 -C18 alkenyl, (C0 -C4 alkyl) CONH2 , (C0 -C4 alkyl) COOH, (C0 -C 4alkyl)NH2 , (C0-C4alkyl )OH and halogen. In another embodiment, R3 is C1 -C6 alkyl, R4 is selected from H and C1 -C4 alkyl, or R3 and R4 together with the atoms to which they are attached form a 5 membered heterocyclic ring . Inone embodiment, Xi is threonine andX3 is asparagine or glycine.
在一个实施方案中，R1选自H和C1-C6烷基，R2选自H、C1-C6烷基、(C1-C4烷基)C(O)NH2、CH2OH、(C1-C4烷基)NH2、(C3-C6环烷基)和CH2(C6芳基)R7，或者R2和R6与它们所连接的原子一起形成5元杂环；In one embodiment, R1 is selected from H and C1 -C6 alkyl, R2 is selected from H, C1 -C6 alkyl, (C1 -C4 alkyl)C(O)NH2 , CH2 OH, (C1 -C4 alkyl) NH2 , (C3 -C6 cycloalkyl) and CH2 (C6 aryl) R7 , or R2 and R6 and the atoms to which they are attached together form a 5-membered heterocycle;
R3为C1-C6烷基；R3 is C1 -C6 alkyl;
R4选自H、C1-C4烷基、(C3-C6)环烷基和(C0-C4烷基)(C6-C10芳基)R7，或者R3和R4与它们所连接的原子一起形成5元杂环；R4 is selected from H, C1 -C4 alkyl, (C3 -C6 ) cycloalkyl and (C0 -C4 alkyl) (C6 -C10 aryl) R7 , or R3 and R4 together with the atoms to which they are attached form a 5-membered heterocyclic ring;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, or R6 and R2together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring; and
R7选自H和OH。R7 is selected from H and OH.
根据一个实施方案，提供单链胰岛素前药类似物，其中人胰岛素B链或其功能类似物的羧基端与人胰岛素A链或其功能类似物的N端共价连接，且其中具有以下通用结构的二肽前药部分在肽的N端或在氨基酸侧链(包括例如在对应于各自天然胰岛素A链或B链的A19、B16或B25位的位置)通过酰胺键共价结合：According to one embodiment, there is provided a single-chain insulin prodrug analogue, wherein the carboxy-terminus of human insulin B chain or a functional analogue thereof is covalently linked to the N-terminus of human insulin A chain or a functional analogue thereof, and wherein has the following general structure The dipeptide prodrug moiety is covalently bonded via an amide bond at the N-terminus of the peptide or at the amino acid side chain (including, for example, at positions corresponding to positions A19, B16 or B25 of the respective native insulin A or B chains):
根据一个实施方案，单链胰岛素类似物包含式：B-P-A的化合物，其中：B表示人胰岛素的B链或者本文公开的B链的功能类似物或前药类似物之一，A表示人胰岛素的A链或者本文公开的A链的功能类似物或前药类似物之一，P表示包括肽接头在内的接头，其使A链与B链共价连接。在一个实施方案中，接头为约5-约18个、或约10-约14个、或约4-约8个或约6个氨基酸的肽接头。在一个实施方案中，B链通过4-12或4-8个氨基酸的肽接头与A链连接。 According to one embodiment, the single-chain insulin analogue comprises a compound of the formula: BPA, wherein: B represents the B chain of human insulin or one of the functional analogs or prodrug analogs of the B chain disclosed herein, and A represents the A of human insulin chain or one of the functional analogs or prodrug analogs of the A chain disclosed herein, P represents a linker including a peptide linker, which covalently links the A chain to the B chain. In one embodiment, the linker is a peptide linker of about 5 to about 18, or about 10 to about 14, or about 4 to about 8, or about 6 amino acids. In one embodiment, the B chain is linked to the A chain by a peptide linker of 4-12 or 4-8 amino acids.
在一个实施方案中，式B-P-A的单链胰岛素前药类似物包含具有序列GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)的A链和含有序列J-X14-X4LCGX5X6LVEALX7LVCGERGFX8(SEQ ID NO：14)的B链序列，其中J为H或包含以下通用结构的二肽：In one embodiment, the single chain insulin prodrug analogue of formula BPA comprises an A chain having the sequence GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) and comprising the sequence JX14 -X4 LCGX5 X6 LVEALX7 LVCGERGFX The B chain sequence of8 (SEQ ID NO: 14), wherein J is H or a dipeptide comprising the following general structure:
其中X14为键或选自以下的1-4个氨基酸的序列：FVNQ(SEQ ID NO：11)、VNQ、NQ和Q。whereinX14 is a bond or a sequence of 1-4 amino acids selected from: FVNQ (SEQ ID NO: 11), VNQ, NQ and Q.
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
其中X选自OH、NHR10和OCH3；其中R10为H或包含以下通用结构的二肽：Wherein X is selected from OH, NHR10 and OCH3 ; wherein R10 is H or a dipeptide comprising the following general structure:
X3为天冬酰胺、甘氨酸、丙氨酸、苏氨酸或丝氨酸；X3 is asparagine, glycine, alanine, threonine or serine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸；X is selected from histidine, aspartic acid, glutamic acid, homocystesulfonic acid and cysteinesulfonic acid;
X7为以下通用结构的氨基酸X7 is an amino acid of the following general structure
其中X12选自OH、OCH3和NHR11，其中R11为H或包含以下通用结构的二肽：Wherein X12 is selected from OH, OCH3 and NHR11 , wherein R11 is H or a dipeptide comprising the following general structure:
X8为以下通用结构的氨基酸X8 is an amino acid of the following general structure
其中X13选自H、OH、OCH3和NHR12，其中R12为H或包含以下通用结构的二肽：Wherein X13 is selected from H, OH, OCH3 and NHR12 , wherein R12 is H or a dipeptide comprising the following general structure:
其中R1选自H和C1-C8烷基；和wherein R is selected from H and C1-C 8alkyl ; and
R2和R4独立选自H、C1-C8烷基、C2-C8烯基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C2-C3烷基)SCH3、(C1-C4烷基)CONH2、(C1-C4烷基)COOH、(C1-C4烷基)NH2、(C1-C4烷基)NHC(NH2+)NH2、(C0-C4烷基)(C3-C6环烷基)、(C0-C4烷基)(C6-C10芳基)R7和CH2(C5-C9杂芳基)；R2 and R4 are independently selected from H, C1 -C8 alkyl, C2 -C8 alkenyl, (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH, (C2 -C3 alkyl)SCH3 , (C1 -C4 alkyl)CONH2 , (C1 -C4 alkyl)COOH, (C1 -C4 alkyl)NH2 , (C1 -C4 alkyl) NHC (NH2+ ) NH2 , (C0 -C4 alkyl) (C3 -C6 cycloalkyl), (C0 -C4 alkyl) (C6 -C10 aryl ) R7 and CH2 (C5 -C9 heteroaryl);
R3选自C1-C8烷基、(C1-C4烷基)OH、(C1-C4烷基)SH、(C1-C4烷基)NH2、(C3-C6)环烷基，或者R4和R3与它们所连接的原子一起形成5或6元杂环；R3 is selected from C1 -C8 alkyl, (C1 -C4 alkyl) OH, (C1 -C4 alkyl) SH, (C1 -C4 alkyl) NH2 , (C3 - C6 ) cycloalkyl, or R4 and R3 form a 5- or 6-membered heterocyclic ring together with the atoms they are connected to;
R5为NHR6或OH；R5 is NHR6 or OH;
R6为H，或者R6和R2与它们所连接的原子一起形成5或6元杂环；和R6 is H, or R6 and R2together with the atoms to which they are attached form a 5- or6- membered heterocyclic ring; and
R7选自H和OH，前提条件是Z、J、R10、R11或R12中的一个并且只有一个为包含以下通用结构的二肽：R7 is selected from H and OH, provided that one of Z, J, R10 , R11 or R12 and only one is a dipeptide comprising the following general structure:
(即只有一个二肽前药元件与胰岛素肽连接)。 (ie only one dipeptide prodrug element is linked to the insulin peptide).
在一个实施方案中，J为包含以下通用结构的二肽：In one embodiment, J is a dipeptide comprising the general structure:
X和X12各自为OH，X13为H，前提条件是如果R4和R3与它们所连接的原子一起形成5元杂环，则R1和R2两者都不为H。在一个备选实施方案中，X12包含式I的二肽，J和X13各自为H，X为OH。在另一个备选实施方案中，X13包含式I的二肽，X和X12各自为OH，J为H。在另一个实施方案中，X包含式I的二肽，J和X13各自为H，X12为OH。在一个实施方案中，B链包含序列J-X9VNQX4LCGX5X6LVEALX7LVCGERGFX8YTPKT(SEQ ID NO：15)或J-X9VNQX4LCGX5X6LVEALX7LVCGERGFX8YTKPT(SEQ ID NO：16)，其中J、X4、X5、X6、X7、X8和X9如紧接的上文中所定义。在另一个实施方案中，R3为C1-C6烷基，R4选自H、C1-C4烷基，或者R3和R4与它们所连接的原子一起形成5元杂环。在又一个实施方案中，X4为组氨酸，X5为丝氨酸，X6为组氨酸。 X andX12 are each OH andX13 is H with theproviso that neither R1 nor R2 is H if R4 andR3 together with the atoms to which they are attached forma5 membered heterocyclic ring. In an alternative embodiment, X12 comprises a dipeptide of formula I, J and X13 are each H, and X is OH. In another alternative embodiment, X13 comprises a dipeptide of formula I, X and X12 are each OH, and J is H. In another embodiment, X comprises a dipeptideof formula I, J and Xare each H, and X is OH. In one embodiment, the B chain comprises the sequence JX9 VNQX4 LCGX5 X6 LVEALX7 LVCGERGFX8 YTPKT (SEQ ID NO: 15) or JX9 VNQX4 LCGX5 X6 LVEALX7 LVCGERGFX8 YTKPT (SEQ ID NO: 16 ), wherein J, X4 , X5 , X6 , X7 , X8 and X9 are as defined immediately above. In another embodiment, R3 is C1 -C6 alkyl, R4 is selected from H, C1 -C4 alkyl, or R3 and R4 together with the atoms to which they are attached form a 5 membered heterocyclic ring .In yet another embodiment,X4 is histidine, X5 is serine, andX6 is histidine.
在一个实施方案中，单链胰岛素类似物包含式：B-P-A的化合物，其中：In one embodiment, the single chain insulin analogue comprises a compound of formula: B-P-A, wherein:
B表示包含序列X4LCGX5X6LVEALYLVCGERGFF(SEQ ID NO：4)的B链序列或其功能类似物，B represents the B chain sequence comprising the sequence X4 LCGX5 X6 LVEALYLVCGERGFF (SEQ ID NO: 4) or a functional analogue thereof,
A表示包含序列GIVEQCCX1SICSLYQLENX2CX3(SEQ ID NO：3)的A链序列或其功能类似物，其中A represents an A chain sequence comprising the sequence GIVEQCCX1 SICSLYQLENX2 CX3 (SEQ ID NO: 3) or a functional analogue thereof, wherein
X1选自苏氨酸和组氨酸；X is selected from threonine and histidine;
X2为以下通用结构的氨基酸X2 is an amino acid of the following general structure
X3为天冬酰胺或甘氨酸；X3 is asparagine or glycine;
X4选自组氨酸和苏氨酸；X is selected from histidine and threonine;
X5选自丙氨酸、甘氨酸和丝氨酸；X is selected from alanine, glycine and serine;
X6选自组氨酸、天冬氨酸、谷氨酸、高半胱磺酸和半胱磺酸。X is selected fromhistidine , aspartic acid, glutamic acid, homocysteic acid and cysteic acid.
P表示包括肽接头在内的接头，其将A链的氨基端与B链的羧基端共价连接。在一个备选实施方案中，单链胰岛素类似物包含式：A-P-B的化合物，其中：A表示人胰岛素A链或其功能类似物，B表示人胰岛素B链或其功能类似物，P表示包括肽接头在内的接头，其将B链的氨基端与A链的羧基端共价连接。在一个实施方案中，肽接头包含4-8个氨基酸。P denotes linkers, including peptide linkers, which covalently link the amino terminus of the A chain to the carboxy terminus of the B chain. In an alternative embodiment, the single chain insulin analog comprises a compound of the formula: A-P-B, wherein: A represents human insulin A chain or a functional analog thereof, B represents human insulin B chain or a functional analog thereof, and P represents a peptide comprising A linker, including a linker, which covalently links the amino terminus of the B chain to the carboxyl terminus of the A chain. In one embodiment, the peptide linker comprises 4-8 amino acids.
根据一个实施方案，肽接头长度为5-18个氨基酸，且包含选自以下的序列：Gly-Gly-Gly-Pro-Gly-Lys-Arg(SEQ ID NO：22)、Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr(SEQ ID NO：23)、Arg-Arg-Gly-Pro-Gly-Gly-Gly(SEQ ID NO：32)、Gly-Gly-Gly-Gly-Gly-Lys-Arg(SEQ ID NO：24)、Arg-Arg-Gly-Gly-Gly-Gly-Gly(SEQ ID NO：25)、Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg(SEQ ID NO：26)、Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly(SEQ ID NO：27)、Gly-Gly-Tyr-Pro-GIy-Asp-Val-Lys-Arg(SEQ ID NO：28)、Arg-Arg-Tyr-Pro-Gly-Asp-Val-Gly-Gly(SEQ ID NO：29)、Gly-Gly-His-Pro-Gly-Asp-Val-Lys-Arg(SEQ ID NO：30)和Arg-Arg-His-Pro-Gly-Asp-Val-Gly-Gly(SEQ ID NO：31)。在一个实施方案中，肽接头的长度为7-12个氨基酸且包含序列Gly-Gly-Gly-Pro-Gly-Lys-Arg(SEQ ID NO：22)或Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr(SEQ ID NO：23)。According to one embodiment, the peptide linker is 5-18 amino acids in length and comprises a sequence selected from the group consisting of: Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 22), Gly-Tyr-Gly- Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr (SEQ ID NO: 23), Arg-Arg-Gly-Pro-Gly-Gly-Gly (SEQ ID NO: 32), Gly-Gly- Gly-Gly-Gly-Lys-Arg (SEQ ID NO: 24), Arg-Arg-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 25), Gly-Gly-Ala-Pro-Gly-Asp- Val-Lys-Arg (SEQ ID NO: 26), Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 27), Gly-Gly-Tyr-Pro-GIy-Asp- Val-Lys-Arg (SEQ ID NO: 28), Arg-Arg-Tyr-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 29), Gly-Gly-His-Pro-Gly-Asp- Val-Lys-Arg (SEQ ID NO: 30) and Arg-Arg-His-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 31). In one embodiment, the peptide linker is 7-12 amino acids in length and comprises the sequence Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 22) or Gly-Tyr-Gly-Ser-Ser- Ser-Arg-Arg-Ala-Pro-Gln-Thr (SEQ ID NO: 23).
在另一个实施方案中，肽接头包含选自以下的序列：AGRGSGK(SEQ ID NO：35)、AGLGSGK(SEQ ID NO：36)、AGMGSGK(SEQ ID NO：37)、ASWGSGK(SEQ ID NO：38)、TGLGSGQ(SEQID NO：39)、TGLGRGK(SEQ ID NO：40)、TGLGSGK(SEQ ID NO：41)、HGLYSGK(SEQ ID NO：42)、KGLGSGQ(SEQ ID NO：43)、VGLMSGK(SEQ ID NO：44)、VGLSSGQ(SEQ ID NO：45)、VGLYSGK(SEQID NO：46)、VGLSSGK(SEQ ID NO：47)、VGMSSGK(SEQ ID NO：48)、VWSSSGK(SEQ ID NO：49)、VGSSSGK(SEQ ID NO：50)、VGMSSGK(SEQ ID NO：51)、TGLGSGR(SEQ ID NO：52)、TGLGKGQ(SEQID NO：53)、KGLSSGQ(SEQ ID NO：54)、VKLSSGQ(SEQ ID NO：55)、VGLKSGQ(SEQ ID NO：56)、TGLGKGQ(SEQ ID NO：57)、SRVSRRSR(SEQ ID NO：65)、GYGSSSRRAPQT(SEQ ID NO：66)和VGLSKGQ(SEQ ID NO：58)。在一个实施方案中，接头包含GSSSRRAP(SEQ ID NO：67)或SRVSRRSR(SEQ ID NO：65)。In another embodiment, the peptide linker comprises a sequence selected from the group consisting of: AGRGSGK (SEQ ID NO: 35), AGLGSGK (SEQ ID NO: 36), AGMGSGK (SEQ ID NO: 37), ASWGSGK (SEQ ID NO: 38 ), TGLGSGQ (SEQ ID NO: 39), TGLGRGK (SEQ ID NO: 40), TGLGSGK (SEQ ID NO: 41), HGLYSGK (SEQ ID NO: 42), KGLGSGQ (SEQ ID NO: 43), VGLMSGK (SEQ ID NO: 44), VGLSSGQ (SEQ ID NO: 45), VGLYSGK (SEQ ID NO: 46), VGLSSGK (SEQ ID NO: 47), VGMSSGK (SEQ ID NO: 48), VWSSSGK (SEQ ID NO: 49), VGSSSGK (SEQ ID NO:50), VGMSSGK (SEQ ID NO:51), TGLGSGR (SEQ ID NO:52), TGLGKGQ (SEQ ID NO:53), KGLSSGQ (SEQ ID NO:54), VKLSSGQ (SEQ ID NO:55 ), VGLKSGQ (SEQ ID NO: 56), TGLGKGQ (SEQ ID NO: 57), SRVSRRSR (SEQ ID NO: 65), GYGSSSRRAPQT (SEQ ID NO: 66), and VGLSKGQ (SEQ ID NO: 58). In one embodiment, the linker comprises GSSSRRAP (SEQ ID NO: 67) or SRVSRRSR (SEQ ID NO: 65).
在一个实施方案中，单链胰岛素类似物具有以下氨基酸序列：Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Xaa-Cys-Asn(SEQ ID NO：33)或Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Xaa-Cys-Asn(SEQ ID NO：34)，其中Xaa为以下通用结构的氨基酸In one embodiment, the single chain insulin analog has the following amino acid sequence: Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu- Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser- Leu-Tyr-Gln-Leu-Glu-Asn-Xaa-Cys-Asn (SEQ ID NO: 33) or Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu -Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser -Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Xaa-Cys-Asn (SEQ ID NO: 34), wherein Xaa is an amino acid of the following general structure
本文公开的胰岛素肽可以为包含通过接头结合的至少2、3或更多个肽的二聚体、三聚体或更高级次的多聚体的部分，其中至少一个或两个肽包含与胰岛素肽连接的二肽前药元件。二聚体可以是同二聚体或异二聚体，包含选自天然胰岛素、天然IGF-1、天然IGF-II和本文公开的胰岛素类似物肽的肽。在一个实施方案中，接头选自双官能巯基交联剂和双官能胺交联剂。在某些实施方案中，接头为PEG，例如5kDa PEG、20kDa PEG。在一些实施方案中，接头为二硫键。The insulin peptides disclosed herein may be part of a dimer, trimer or higher order multimer comprising at least 2, 3 or more peptides bound by a linker, wherein at least one or two peptides comprise Peptide linked dipeptide prodrug elements. The dimer may be a homodimer or a heterodimer comprising a peptide selected from native insulin, native IGF-1, native IGF-II and the insulin analog peptides disclosed herein. In one embodiment, the linker is selected from difunctional thiol crosslinkers and difunctional amine crosslinkers. In certain embodiments, the linker is PEG, eg, 5kDa PEG, 20kDa PEG. In some embodiments, the linker is a disulfide bond.
例如，二聚体的每个单体可包含Cys残基(例如位于末端的Cys或位于内部的Cys)，且每个Cys残基的硫原子参与二硫键的形成。二聚体的每个单体代表A链和B链的异二聚体。A链和B链通过二硫键连接，或制备成单链肽。在本发明的一些方面，单体通过末端氨基酸(例如N端或C端)、通过内部氨基酸或通过至少一个单体的末端氨基酸和至少一个其它单体的内部氨基酸连接起来。在具体的方面，单体不通过N端氨基酸连接。在一些方面，多聚体的单体以“尾对尾(tail-to-tail)”方向连接在一起，其中每个单体的C端氨基酸连接在一起。缀合物部分可与本文所述的任何胰岛素肽(包括二聚体、三聚体或更高级次多聚体)共价连接。For example, each monomer of the dimer may comprise a Cys residue (eg, a terminal Cys or an internal Cys), and the sulfur atom of each Cys residue participates in the formation of a disulfide bond. Each monomer of the dimer represents a heterodimer of A chain and B chain. Chain A and B are linked by a disulfide bond, or prepared as a single-chain peptide. In some aspects of the invention, monomers are linked via a terminal amino acid (eg, N-terminal or C-terminal), via an internal amino acid, or via the terminal amino acid of at least one monomer and the internal amino acid of at least one other monomer. In specific aspects, the monomers are not linked through the N-terminal amino acid. In some aspects, the monomers of the multimer are linked together in a "tail-to-tail" orientation, wherein the C-terminal amino acids of each monomer are linked together. The conjugate moiety can be covalently linked to any of the insulin peptides described herein, including dimers, trimers or higher order multimers.
还可修饰本文公开的前药以改进肽在生理pH下肽在水性溶液中的溶解度，同时通过防止肽的肾清除而提高肽的有效持续时间。因为与血浆蛋白质相比，肽的分子大小相对较小，因此肽容易被清除掉。将肽的分子量增加到40kDa以上超过了肾阈，从而显著延长了在血浆中的持续时间。因此，在一个实施方案中，对肽前药作进一步修饰以包含共价连接的亲水部分。在一个实施方案中，亲水部分为血浆蛋白聚环氧乙烷链或免疫球蛋白Fc部分。因此，在一个实施方案中，对本发明公开的前药作进一步修饰以包含一个或多个与氨基酸的侧链共价连接的亲水基团。The prodrugs disclosed herein can also be modified to improve the solubility of the peptide in aqueous solution at physiological pH, while increasing the effective duration of the peptide by preventing renal clearance of the peptide. Peptides are easily cleared because of their relatively small molecular size compared to plasma proteins. Increasing the molecular weight of the peptide above 40 kDa crosses the renal threshold, thereby significantly prolonging the duration in plasma. Thus, in one embodiment, the peptide prodrug is further modified to include a covalently attached hydrophilic moiety. In one embodiment, the hydrophilic portion is a plasma protein polyethylene oxide chain or an immunoglobulin Fc portion. Accordingly, in one embodiment, the prodrugs disclosed herein are further modified to include one or more hydrophilic groups covalently attached to the side chains of the amino acids.
根据一个实施方案，通过将亲水部分与B链的N端氨基酸或位于B链羧基端的赖氨酸氨基酸(包括例如SEQ ID NO：9/SEQ ID NO：13的28位或SEQ ID NO：8/SEQ ID NO：12的29位)的侧链连接，来进一步修饰本文公开的胰岛素前药。在一个实施方案中，提供单链胰岛素前药类似物，其中通过将亲水部分与肽接头的侧链连接来修饰肽接头的一个氨基酸。在一个实施方案中，修饰氨基酸为半胱氨酸、赖氨酸或乙酰基苯丙氨酸。在一个实施方案中，肽接头选自TGLGSGQ(SEQ ID NO：39)、VGLSSGQ(SEQ ID NO：45)、VGLSSGK(SEQ ID NO：47)、TGLGSGR(SEQ ID NO：52)、TGLGKGQ(SEQ ID NO：53)、KGLSSGQ(SEQ ID NO：54)、VKLSSGQ(SEQID NO：55)、VGLKSGQ(SEQ ID NO：56)、TGLGKGQ(SEQ ID NO：57)和VGLSKGQ(SEQ ID NO：58)，且亲水部分(例如聚乙二醇)与肽接头的赖氨酸侧链连接。According to one embodiment, by combining the hydrophilic part with the N-terminal amino acid of the B chain or the lysine amino acid located at the carboxy-terminal of the B chain (including, for example, the 28th position of SEQ ID NO: 9/SEQ ID NO: 13 or SEQ ID NO: 8 /SEQ ID NO: 29 of 12) to further modify the insulin prodrug disclosed herein. In one embodiment, a single chain insulin prodrug analog is provided wherein one amino acid of the peptide linker is modified by attaching a hydrophilic moiety to the side chain of the peptide linker. In one embodiment, the modified amino acid is cysteine, lysine or acetylphenylalanine. In one embodiment, the peptide linker is selected from TGLGSGQ (SEQ ID NO: 39), VGLSSGQ (SEQ ID NO: 45), VGLSSGK (SEQ ID NO: 47), TGLGSGR (SEQ ID NO: 52), TGLGKGQ (SEQ ID NO: 53), KGLSSGQ (SEQ ID NO: 54), VKLSSGQ (SEQ ID NO: 55), VGLKSGQ (SEQ ID NO: 56), TGLGKGQ (SEQ ID NO: 57) and VGLSKGQ (SEQ ID NO: 58), and A hydrophilic moiety (eg, polyethylene glycol) is attached to the lysine side chain of the peptide linker.
在另一个实施方案中，通过将修饰氨基酸加至胰岛素前药B链的羧基端，来进一步修饰本文公开的胰岛素前药类似物，其中对C端加入的氨基酸进行修饰以包含与该氨基酸连接的亲水部分。在一个实施方案中，加到C端的氨基酸为修饰的半胱氨酸、赖氨酸或乙酰基苯丙氨酸。在一个实施方案中，亲水部分选自血浆蛋白、聚环氧乙烷链和免疫球蛋白的Fc部分。In another embodiment, the insulin prodrug analogs disclosed herein are further modified by adding a modified amino acid to the carboxyl-terminus of the B-chain of the insulin prodrug, wherein the amino acid added at the C-terminus is modified to include a Hydrophilic part. In one embodiment, the amino acid added to the C-terminus is a modified cysteine, lysine or acetylphenylalanine. In one embodiment, the hydrophilic portion is selected from plasma proteins, polyethylene oxide chains and Fc portions of immunoglobulins.
在一个实施方案中，亲水基团为聚环氧乙烷链，而在一个实施方案中，两个或更多个聚环氧乙烷链与胰岛素前药类似物的两个或更多个氨基酸侧链共价连接。根据一个实施方案，亲水部分在选自以下的位置与本文公开的胰岛素前药类似物的氨基酸侧链共价连接：A9、A14、A15、B22、B28、B29和B链的C端或N端。对于具有多个聚环氧乙烷链的胰岛素前药类似物，聚环氧乙烷链可以在B链的N端氨基酸上连接，或者与位于B链羧基端的赖氨酸氨基酸的侧链连接，或者通过在肽的C端添加单个氨基酸而连接，其中所添加的氨基酸具有与其侧链连接的聚环氧乙烷链。根据一个实施方案，聚环氧乙烷链或其它亲水部分与包含二肽前药元件的两个氨基酸之一的侧链连接。在一个实施方案中，二肽前药元件包含具有与其侧链胺连接的聚环氧乙烷链的赖氨酸(D或L构型)。In one embodiment, the hydrophilic groups are polyethylene oxide chains, and in one embodiment, two or more polyethylene oxide chains are combined with two or more Amino acid side chains are covalently linked. According to one embodiment, the hydrophilic moiety is covalently linked to the amino acid side chain of the insulin prodrug analog disclosed herein at a position selected from: A9, A14, A15, B22, B28, B29 and the C-terminus or N of the B chain. end. For insulin prodrug analogs with multiple polyethylene oxide chains, the polyethylene oxide chain can be attached to the N-terminal amino acid of the B chain, or to the side chain of the lysine amino acid located at the carboxy-terminal of the B chain, Or by adding a single amino acid at the C-terminus of the peptide, where the added amino acid has a polyethylene oxide chain attached to its side chain. According to one embodiment, a polyethylene oxide chain or other hydrophilic moiety is attached to the side chain of one of the two amino acids comprising the dipeptide prodrug element. In one embodiment, the dipeptide prodrug element comprises a lysine (D or L configuration) with a polyethylene oxide chain attached to its side chain amine.
亲水部分的连接Hydrophilic link
在另一个实施方案中，通过将亲水部分与肽共价连接来提高本文公开的胰岛素类似物的溶解度。可在任何用于使蛋白质与活化聚合物分子反应的合适条件下，将亲水部分与胰岛素类似物连接。可采用本领域已知的任何方法，包括通过酰化、还原烷基化、迈克尔加成(Michael addition)、巯基烷基化或通过PEG部分上的反应基(例如醛、氨基、酯、巯基、α-卤代乙酰基、马来酰亚胺基或肼基)与目标化合物上的反应基(例如醛、氨基、酯、巯基、α-卤代乙酰基、马来酰亚胺基或肼基)进行的其它化学选择性缀合/连接方法。可用于将水溶性聚合物与一个或多个蛋白质连接的活化基团包括而不限于砜、马来酰亚胺、硫氢基、巯基、三氟甲磺酸基、三氟乙磺酸基、氮丙烯(azidirine)、环氧乙烷和5-吡啶基。如果通过还原烷基化与肽连接，则所选择的聚合物应具有单一活性醛，使得聚合程度受到控制。参见例如Kinstler等，Adv.Drug.Delivery Rev.54：477-485(2002)；Roberts等，Adv.DrugDelivery Rev.54：459-476(2002)；以及Zalipsky等，Adv.Drug.Delivery Rev.16：157-182(1995)。In another embodiment, the solubility of the insulin analogs disclosed herein is increased by covalently linking a hydrophilic moiety to the peptide. Attachment of the hydrophilic moiety to the insulin analog can be done under any suitable conditions for reacting the protein with the activated polymer molecule. Any method known in the art may be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation, or via reactive groups on the PEG moiety (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimido, or hydrazino) with reactive groups on target compounds such as aldehydes, amino groups, esters, thiols, α-haloacetyl, maleimido, or hydrazinos ) other chemoselective conjugation/linkage methods. Activating groups that can be used to attach a water soluble polymer to one or more proteins include, without limitation, sulfone, maleimide, sulfhydryl, sulfhydryl, triflate, trifluoroethanesulfonate, Azidirine, oxirane and 5-pyridyl. If linking to the peptide is via reductive alkylation, the selected polymer should have a single reactive aldehyde such that the degree of polymerization is controlled. See, eg, Kinstler et al., Adv. Drug. Delivery Rev. 54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476 (2002); and Zalipsky et al., Adv. Drug. Delivery Rev. 16 : 157-182 (1995).
合适的亲水部分包括聚乙二醇(PEG)、聚丙二醇、聚氧乙烯化多元醇(例如POG)、聚氧乙烯化山梨醇、聚氧乙烯化葡萄糖、聚氧乙烯化甘油(POG)、聚氧化烯、聚乙二醇丙醛、乙二醇/丙二醇共聚物、聚乙二醇单甲醚、一-(C1-C10)烷氧基-聚乙二醇或芳氧基-聚乙二醇、羧甲基纤维素、聚缩醛、聚乙烯醇(PVA)、聚乙烯吡咯烷酮、聚-1，3-二氧杂环戊烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚(β-氨基酸)(均聚物或无规共聚物)、聚(n-乙烯基吡咯烷酮)聚乙二醇、聚丙二醇均聚物(PPG)和其它聚环氧烷、聚环氧丙烷/环氧乙烷共聚物、肠菌酸(colonic acid)或其它多糖聚合物、菲可(Ficoll)或葡聚糖及其混合物。Suitable hydrophilic moieties include polyethylene glycol (PEG), polypropylene glycol, polyoxyethylated polyols (such as POG), polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), Polyoxyalkylene, polyethylene glycol propionaldehyde, ethylene glycol/propylene glycol copolymer, polyethylene glycol monomethyl ether, mono-(C1-C10)alkoxy-polyethylene glycol or aryloxy-polyethylene glycol Alcohol, carboxymethylcellulose, polyacetal, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-tri alkanes, ethylene/maleic anhydride copolymers, poly(β-amino acids) (homopolymers or random copolymers), poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer (PPG) and others Polyalkylene oxide, polypropylene oxide/ethylene oxide copolymer, colonic acid or other polysaccharide polymers, Ficoll or dextran and mixtures thereof.
根据一个实施方案，亲水部分(例如聚乙二醇链)的分子量选自约500-约40,000道尔顿。在一个实施方案中，亲水部分(例如PEG)的分子量选自约500-约5,000道尔顿、或约1,000-约5,000道尔顿。在另一个实施方案中，亲水部分(例如PEG)的分子量为约10,000-约20,000道尔顿。在其它示例性的实施方案中，亲水部分(例如PEG)的分子量为约20,000-约40,000道尔顿。According to one embodiment, the molecular weight of the hydrophilic portion (eg polyethylene glycol chain) is selected from about 500 to about 40,000 Daltons. In one embodiment, the molecular weight of the hydrophilic moiety (eg, PEG) is selected from about 500 to about 5,000 Daltons, or about 1,000 to about 5,000 Daltons. In another embodiment, the molecular weight of the hydrophilic moiety (eg, PEG) is from about 10,000 to about 20,000 Daltons. In other exemplary embodiments, the molecular weight of the hydrophilic moiety (eg, PEG) is from about 20,000 to about 40,000 Daltons.
在一个实施方案中，将葡聚糖用作亲水部分。葡聚糖是主要通过α1-6连接的葡萄糖亚基的多糖聚合物。可得到许多分子量范围的葡聚糖，例如约1kD-约100kD，或约5、10、15或20kD至约20、30、40、50、60、70、80或90kD。In one embodiment, dextran is used as the hydrophilic moiety. Dextran is a polysaccharide polymer of glucose subunits linked primarily by α1-6. Dextran is available in a number of molecular weight ranges, for example from about 1 kD to about 100 kD, or from about 5, 10, 15 or 20 kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD.
考虑了直链或支链聚合物。缀合物的所得制备物可以是基本单分散性的或多分散性的，且可具有每个肽约0.5、0.7、1、1.2、1.5或2个聚合物部分。Linear or branched polymers are contemplated. The resulting preparations of conjugates may be substantially monodisperse or polydisperse, and may have about 0.5, 0.7, 1, 1.2, 1.5 or 2 polymer moieties per peptide.
根据一个实施方案，本文公开的胰岛素前药类似物被氨基酸取代进一步修饰，其中取代氨基酸包含适于与亲水部分(包括例如聚乙二醇)交联的侧链。在一个实施方案中，在待亲水部分连接的胰岛素前药类似物位置上的氨基酸被天然或合成氨基酸取代(或加在C端上)，以引入亲水部分或使亲水部分容易连接。例如，在一个实施方案中，在选自A5、A8、A9、A10、A12、A14、A15、A17、A18、B1、B2、B3、B4、B5、B13、B14、B17、B21、B22、B26、B27、B28、B29和B30的位置上的天然氨基酸被赖氨酸、半胱氨酸或乙酰基苯丙氨酸残基取代(或将赖氨酸、半胱氨酸或乙酰基苯丙氨酸残基加在C端上)，以供聚乙二醇链共价连接。According to one embodiment, the insulin prodrug analogs disclosed herein are further modified by amino acid substitutions, wherein the substituted amino acids comprise side chains suitable for cross-linking with hydrophilic moieties including, for example, polyethylene glycol. In one embodiment, the amino acid at the position of the insulin prodrug analog to which the hydrophilic moiety is to be attached is substituted with a natural or synthetic amino acid (or added at the C-terminus) to introduce or facilitate attachment of the hydrophilic moiety. For example, in one embodiment, in the group selected from A5, A8, A9, A10, A12, A14, A15, A17, A18, B1, B2, B3, B4, B5, B13, B14, B17, B21, B22, B26 , B27, B28, B29 and B30 positions of natural amino acids are replaced by lysine, cysteine or acetylphenylalanine residues (or lysine, cysteine or acetylphenylalanine Acid residues are added at the C-terminus) for covalent attachment of polyethylene glycol chains.
在一个实施方案中，胰岛素前药类似物具有单个加在B链的羧基端上的半胱氨酸残基，或者胰岛素前药类似物被至少一个半胱氨酸残基取代，其中将所述半胱氨酸残基的侧链用巯基反应性试剂进一步修饰，所述巯基反应性试剂包括例如马来酰亚胺基、乙烯砜、2-吡啶基巯基、卤代烷基和卤代酰基。这些巯基反应性试剂可含有羧基、酮基、羟基和醚基团以及其它亲水部分，例如聚乙二醇单元。在一个备选实施方案中，胰岛素前药类似物具有单个加到B链的羧基端上的赖氨酸残基，或者胰岛素前药类似物被赖氨酸取代，以及使用胺反应性试剂例如羧酸的活性酯(琥珀酰亚胺基、酐等)或亲水部分(例如聚乙二醇)的醛对取代赖氨酸残基的侧链进行进一步修饰。In one embodiment, the insulin prodrug analog has a single cysteine residue added to the carboxy terminus of the B chain, or the insulin prodrug analog is substituted with at least one cysteine residue, wherein the The side chains of cysteine residues are further modified with sulfhydryl-reactive reagents including, for example, maleimide, vinylsulfone, 2-pyridylsulfhydryl, haloalkyl, and haloacyl groups. These sulfhydryl-reactive reagents may contain carboxyl, keto, hydroxyl and ether groups as well as other hydrophilic moieties such as polyethylene glycol units. In an alternative embodiment, the insulin prodrug analog has a single lysine residue added to the carboxyl terminus of the B chain, or the insulin prodrug analog is substituted with lysine, and the use of an amine reactive reagent such as carboxyl The side chains of substituted lysine residues are further modified by active esters of acids (succinimide groups, anhydrides, etc.) or aldehydes of hydrophilic moieties (eg polyethylene glycol).
在其中胰岛素前药类似物包含聚乙二醇链的实施方案中，聚乙二醇链可呈直链的形式，或者其可以是支链的。根据一个实施方案，聚乙二醇链的平均分子量为约20,000-约60,000道尔顿。多条聚乙二醇链可与胰岛素前药类似物连接以提供具有最佳溶解度和血液清除特征的胰岛素前药类似物。在一个实施方案中，胰岛素前药类似物与平均分子量为约20,000-约60,000道尔顿的单一聚乙二醇链连接。在另一个实施方案中，胰岛素前药类似物与两条聚乙二醇链连接，其中两条链总的平均分子量为约40,000-约80,000道尔顿。在一个实施方案中，平均分子量为20,000或60,000道尔顿的单一聚乙二醇链与胰岛素前药类似物连接。在另一个实施方案中，单一聚乙二醇链与胰岛素前药类似物连接，且平均分子量为约40,000-约50,000道尔顿。在一个实施方案中，两条聚乙二醇链与胰岛素前药类似物连接，其中第一条和第二条聚乙二醇链各自的平均分子量为20,000道尔顿。在另一个实施方案中，两条聚乙二醇链与胰岛素前药类似物连接，其中第一条和第二条聚乙二醇链各自的平均分子量为40,000道尔顿。In embodiments wherein the insulin prodrug analog comprises a polyethylene glycol chain, the polyethylene glycol chain may be in the form of a linear chain, or it may be branched. According to one embodiment, the average molecular weight of the polyethylene glycol chains is from about 20,000 to about 60,000 Daltons. Multiple polyethylene glycol chains can be attached to the insulin prodrug analog to provide an insulin prodrug analog with optimal solubility and blood clearance characteristics. In one embodiment, the insulin prodrug analog is linked to a single polyethylene glycol chain having an average molecular weight of about 20,000 to about 60,000 Daltons. In another embodiment, the insulin prodrug analog is linked to two polyethylene glycol chains, wherein the combined average molecular weight of the two chains is from about 40,000 to about 80,000 Daltons. In one embodiment, a single polyethylene glycol chain with an average molecular weight of 20,000 or 60,000 Daltons is attached to the insulin prodrug analog. In another embodiment, a single polyethylene glycol chain is attached to the insulin prodrug analog and has an average molecular weight of about 40,000 to about 50,000 Daltons. In one embodiment, two polyethylene glycol chains are linked to the insulin prodrug analog, wherein the first and second polyethylene glycol chains each have an average molecular weight of 20,000 Daltons. In another embodiment, two polyethylene glycol chains are linked to the insulin prodrug analog, wherein the first and second polyethylene glycol chains each have an average molecular weight of 40,000 Daltons.
在另一个实施方案中，提供包含与肽共价结合的两条或更多条聚乙二醇链的胰岛素前药类似物，其中聚乙二醇链的总分子量为约40,000-约60,000道尔顿。在一个实施方案中，聚乙二醇化胰岛素前药类似物包含与选自B链N端的一个或多个氨基酸和/或SEQ IDNO：9的28位或SEQ ID NO：8的29位连接的聚乙二醇链，其中PEG链总的分子量为约40,000-约80,000道尔顿。In another embodiment, there is provided an insulin prodrug analog comprising two or more polyethylene glycol chains covalently bound to a peptide, wherein the total molecular weight of the polyethylene glycol chains is from about 40,000 to about 60,000 Dal pause. In one embodiment, the PEGylated insulin prodrug analogue comprises a polyamino acid linked to one or more amino acids selected from the N-terminus of the B chain and/or the 28th position of SEQ ID NO:9 or the 29th position of SEQ ID NO:8. Ethylene glycol chains, wherein the total molecular weight of the PEG chains is from about 40,000 to about 80,000 Daltons.
根据一个实施方案，胰岛素肽或其前药/长效衍生物与辅助肽融合，所述辅助肽能够形成类似于化学性PEG(例如重组PEG(rPEG)分子)的延伸构象，例如国际专利申请公开号WO2009/023270和美国专利申请公开号US2008/0286808中披露的辅助肽。rPEG分子不是聚乙二醇。在一些方面，rPEG分子是包含一个或多个甘氨酸、丝氨酸、谷氨酸、天冬氨酸、丙氨酸或脯氨酸的多肽。在一些方面，rPEG是均聚物，例如聚甘氨酸、聚丝氨酸、聚谷氨酸、聚天冬氨酸、聚丙氨酸或聚脯氨酸。在其它实施方案中，rPEG包含重复的两种氨基酸，例如聚(Gly-Ser)、聚(Gly-Glu)、聚(Gly-A1a)、聚(Gly-Asp)、聚(Gly-Pro)、聚(Ser-Glu)等。在一些方面，rPEG包含三种不同类型的氨基酸，例如聚(Gly-Ser-Glu)。在具体的方面，rPEG延长胰岛素肽的半寿期。在一些方面，rPEG包含净的正电荷或净的负电荷。在一些方面，rPEG缺乏二级结构。在一个实施方案中，rPEG的长度大于或等于10个氨基酸，而在一个实施方案中，长度为约40-约50个氨基酸。在一些方面，辅助肽通过肽键或蛋白酶切割位点与本发明肽的N端或C端融合，或者插入本发明肽的环中。在一些方面，rPEG包含亲和标签或与大于5kDa的PEG连接。在一个实施方案中，rPEG赋予本发明肽增加的流体动力学半径、血清半寿期、蛋白酶抗性或溶解度，而在一些方面赋予肽降低的免疫原性。According to one embodiment, the insulin peptide or its prodrug/long-acting derivative is fused to a helper peptide capable of forming an extended conformation similar to a chemical PEG (e.g. recombinant PEG (rPEG) molecule), e.g. International Patent Application Publication Helper peptides disclosed in WO2009/023270 and US Patent Application Publication No. US2008/0286808. The rPEG molecule is not polyethylene glycol. In some aspects, the rPEG molecule is a polypeptide comprising one or more of glycine, serine, glutamic acid, aspartic acid, alanine, or proline. In some aspects, rPEG is a homopolymer, such as polyglycine, polyserine, polyglutamic acid, polyaspartic acid, polyalanine, or polyproline. In other embodiments, rPEG comprises repeats of two amino acids, such as poly(Gly-Ser), poly(Gly-Glu), poly(Gly-A1a), poly(Gly-Asp), poly(Gly-Pro), Poly(Ser-Glu) et al. In some aspects, rPEG comprises three different types of amino acids, eg, poly(Gly-Ser-Glu). In a specific aspect, rPEG extends the half-life of the insulin peptide. In some aspects, rPEG comprises a net positive charge or a net negative charge. In some aspects, rPEG lacks secondary structure. In one embodiment, the rPEG is greater than or equal to 10 amino acids in length, and in one embodiment, is about 40 to about 50 amino acids in length. In some aspects, a helper peptide is fused to the N- or C-terminus of a peptide of the invention via a peptide bond or a protease cleavage site, or inserted into a loop of a peptide of the invention. In some aspects, the rPEG comprises an affinity tag or is linked to a PEG greater than 5 kDa. In one embodiment, rPEG confers increased hydrodynamic radius, serum half-life, protease resistance or solubility, and in some aspects reduced immunogenicity, to the peptides of the invention.
根据一个实施方案，提供胰岛素前药类似物，其中血浆蛋白与该肽的氨基酸侧链共价连接以改进胰岛素前药类似物的溶解度、稳定性和/或药代动力学。例如，血清白蛋白可与本文提供的胰岛素前药类似物共价结合。在一个实施方案中，血浆蛋白与B链的N端和/或与SEQ ID NO：9的28位或SEQ ID NO：8的29位对应的氨基酸共价结合。According to one embodiment, an insulin prodrug analog is provided wherein a plasma protein is covalently linked to the amino acid side chain of the peptide to improve the solubility, stability and/or pharmacokinetics of the insulin prodrug analog. For example, serum albumin can be covalently bound to the insulin prodrug analogs provided herein. In one embodiment, the plasma protein is covalently bound to the N-terminus of the B chain and/or to the amino acid corresponding to position 28 of SEQ ID NO:9 or position 29 of SEQ ID NO:8.
根据一个实施方案，提供胰岛素前药类似物，其中代表免疫球蛋白分子的Fc部分的线性氨基酸序列与本文公开的胰岛素前药类似物的氨基酸侧链共价连接，以改进胰岛素前药类似物的溶解度、稳定性和/或药代动力学。例如，代表免疫球蛋白分子的Fc部分的氨基酸序列可与B链的N端、或者A链或B链的C端、或者已末端延伸的A链或B链的C端共价结合。例如，代表免疫球蛋白分子的Fc部分的氨基酸序列可与B链的C端共价结合，包括例如与相当于SEQ ID NO：9的28位或SEQ ID NO：8的29位的氨基酸连接。Fc部分通常自IgG中分离，但是来自任何免疫球蛋白的Fc肽片段应起等同作用。According to one embodiment, there is provided an insulin prodrug analog wherein a linear amino acid sequence representing the Fc portion of an immunoglobulin molecule is covalently linked to the amino acid side chains of the insulin prodrug analog disclosed herein to improve the performance of the insulin prodrug analog. Solubility, Stability and/or Pharmacokinetics. For example, an amino acid sequence representing the Fc portion of an immunoglobulin molecule can be covalently bound to the N-terminus of the B chain, or the C-terminus of the A or B chain, or the C-terminus of the terminally extended A or B chain. For example, an amino acid sequence representing the Fc portion of an immunoglobulin molecule may be covalently bound to the C-terminus of the B chain, including, for example, to an amino acid corresponding to position 28 of SEQ ID NO:9 or position 29 of SEQ ID NO:8. The Fc portion is usually isolated from IgG, but Fc peptide fragments from any immunoglobulin should function equally.
在本发明的一个具体方面，通过对胰岛素前药类似物的氨基酸侧链的胺、羟基或巯基进行直接烷基化或酰化，来对胰岛素前药类似物进行修饰以包含烷基或酰基。在一个实施方案中，通过氨基酸的侧链胺、羟基或巯基对胰岛素前药类似物进行直接酰化。在一个实施方案中，在选自以下的一个或多个位置上进行酰化：A9、A14、A15、B22、B28或B29。在这点上，酰化胰岛素前药类似物可包含SEQ ID NO：3的A链氨基酸序列和SEQ ID NO：5的B链，或者SEQ ID NO：3和/或SEQ ID NO：5的修饰氨基酸序列，其中A9、A14、A15、B22、B28或B29位上的至少一个氨基酸被修饰成为包含侧链胺、羟基或巯基的任何氨基酸。在一些具体的实施方案中，通过B28或B29位氨基酸的侧链胺、羟基或巯基进行胰岛素前药类似物的直接酰化。在又一个的实施方案中，胰岛素前药类似物包含与存在于B28或B29位上的Lys的ε-氨基结合并具有1-24个碳原子的羧酸的酰基。在一个实施方案中，提供单链胰岛素前药类似物，其中通过对肽接头的氨基酸侧链的胺、羟基或巯基进行直接酰化，来修饰肽接头的氨基酸之一以包含酰基。根据一个实施方案，单链胰岛素类似物的肽接头选自AGRGSGK(SEQ IDNO：35)、AGLGSGK(SEQ ID NO：36)、AGMGSGK(SEQ ID NO：37)、ASWGSGK(SEQ ID NO：38)、TGLGSGQ(SEQ ID NO：39)、TGLGRGK(SEQ ID NO：40)、TGLGSGK(SEQ ID NO：41)、HGLYSGK(SEQID NO：42)、KGLGSGQ(SEQ ID NO：43)、VGLMSGK(SEQ ID NO：44)、VGLSSGQ(SEQ ID NO：45)、VGLYSGK(SEQ ID NO：46)、VGLSSGK(SEQ ID NO：47)、VGMSSGK(SEQ ID NO：48)、VWSSSGK(SEQID NO：49)、VGSSSGK(SEQ ID NO：50)、VGMSSGK(SEQ ID NO：51)、TGLGSGR(SEQ ID NO：52)、TGLGKGQ(SEQ ID NO：53)、KGLSSGQ(SEQ ID NO：54)、VKLSSGQ(SEQ ID NO：55)、VGLKSGQ(SEQID NO：56)、TGLGKGQ(SEQ ID NO：57)和VGLSKGQ(SEQ ID NO：58)，其中A链中、B链中或连接肽中的至少一个赖氨酸残基通过酰化被化学修饰。在一个实施方案中，酰化基团包含1-5个、10-12个或12-24个碳的链。In a specific aspect of the invention, the insulin prodrug analogs are modified to contain alkyl or acyl groups by direct alkylation or acylation of the amine, hydroxyl or sulfhydryl groups of the amino acid side chains of the insulin prodrug analogs. In one embodiment, the insulin prodrug analog is directly acylated via the side chain amine, hydroxyl or thiol of an amino acid. In one embodiment, the acylation is at one or more positions selected from A9, A14, A15, B22, B28 or B29. In this regard, the acylated insulin prodrug analogue may comprise the amino acid sequence of the A chain of SEQ ID NO: 3 and the B chain of SEQ ID NO: 5, or a modification of SEQ ID NO: 3 and/or SEQ ID NO: 5 Amino acid sequence, wherein at least one amino acid at position A9, A14, A15, B22, B28 or B29 is modified to any amino acid containing side chain amine, hydroxyl or thiol. In some specific embodiments, direct acylation of the insulin prodrug analog is performed through the side chain amine, hydroxyl or thiol of the amino acid at position B28 or B29. In yet another embodiment, the insulin prodrug analogue comprises an acyl group of a carboxylic acid having 1-24 carbon atoms bonded to the ε-amino group of Lys present at position B28 or B29. In one embodiment, a single chain insulin prodrug analog is provided wherein one of the amino acids of the peptide linker is modified to contain an acyl group by direct acylation of an amine, hydroxyl or sulfhydryl group of an amino acid side chain of the peptide linker. According to one embodiment, the peptide linker of the single chain insulin analog is selected from the group consisting of AGRGSGK (SEQ ID NO: 35), AGLGSGK (SEQ ID NO: 36), AGMGSGK (SEQ ID NO: 37), ASWGSGK (SEQ ID NO: 38), TGLGSGQ (SEQ ID NO: 39), TGLGRGK (SEQ ID NO: 40), TGLGSGK (SEQ ID NO: 41), HGLYSGK (SEQ ID NO: 42), KGLGSGQ (SEQ ID NO: 43), VGLMSGK (SEQ ID NO: 44), VGLSSGQ (SEQ ID NO: 45), VGLYSGK (SEQ ID NO: 46), VGLSSGK (SEQ ID NO: 47), VGMSSGK (SEQ ID NO: 48), VWSSSGK (SEQ ID NO: 49), VGSSSGK (SEQ ID NO: 50), VGMSSGK (SEQ ID NO: 51), TGLGSGR (SEQ ID NO: 52), TGLGKGQ (SEQ ID NO: 53), KGLSSGQ (SEQ ID NO: 54), VKLSSGQ (SEQ ID NO: 55) , VGLKSGQ (SEQ ID NO: 56), TGLGKGQ (SEQ ID NO: 57) and VGLSKGQ (SEQ ID NO: 58), wherein at least one lysine residue in the A chain, in the B chain or in the connecting peptide is acylated chemically modified. In one embodiment, the acylation group comprises a chain of 1-5, 10-12, or 12-24 carbons.
根据一个实施方案，进一步修饰本文公开的胰岛素前药类似物以将其它化合物与该类似物的前药二肽部分连接。在一个实施方案中，将包含二肽前药元件的氨基酸侧链聚乙二醇化、酰化或烷基化。在一个实施方案中，二肽用包含1-5个、10-12个或12-24个碳的链的基团酰化。在一个实施方案中，二肽用40-80KDa聚乙二醇链进行聚乙二醇化。在一个实施方案中，二肽前药元件是聚乙二醇化的，且与二肽连接的胰岛素肽是酰化的，包括例如B链C端赖氨酸的酰化。根据一个实施方案，亲水部分或螯合大分子与包含以下通用结构的二肽的R2侧链共价连接：According to one embodiment, the insulin prodrug analogs disclosed herein are further modified to link other compounds to the prodrug dipeptide portion of the analog. In one embodiment, the amino acid side chain comprising the dipeptide prodrug element is pegylated, acylated or alkylated. In one embodiment, the dipeptide is acylated with a group comprising a chain of 1-5, 10-12, or 12-24 carbons. In one embodiment, the dipeptide is pegylated with a 40-80 KDa polyethylene glycol chain. In one embodiment, the dipeptide prodrug element is pegylated and the insulin peptide linked to the dipeptide is acylated, including, for example, acylation of the C-terminal lysine of the B chain. According to one embodiment, a hydrophilic moiety or a chelating macromolecule is covalently linked to the R2 side chain of a dipeptide comprising the following general structure:
其中R2选自(C1-C4烷基)OH、(C1-C4烷基)SH和(C1-C4烷基)NH2。在一个实施方案中，R2为(C3-C4烷基)NH2。螯合大分子为本领域技术人员所知，包括葡聚糖和大分子量聚乙二醇(即大于或等于80KDa)。通过将螯合大分子与二肽部分连接，前药将保持螯合，同时活性胰岛素肽根据二肽酰胺键裂解的动力学慢慢释放。 wherein R2 is selected from (C1 -C4 alkyl)OH, (C1 -C4 alkyl)SH and (C1 -C4 alkyl)NH2 . In one embodiment, R2 is (C3 -C4 alkyl)NH2 . Chelating macromolecules are known to those skilled in the art and include dextran and high molecular weight polyethylene glycols (ie greater than or equal to 80KDa). By linking the chelating macromolecule to the dipeptide moiety, the prodrug will remain chelated while the active insulin peptide is slowly released according to the kinetics of cleavage of the dipeptide amide bond.
本公开内容还包括其它缀合物，其中本发明的胰岛素前药类似物任选通过共价结合和任选通过接头与缀合物连接。可通过共价化学键；诸如静电、氢、离子、范德瓦尔斯或者疏水或亲水相互作用等物理力实现连接。可采用各种非共价偶联系统，包括生物素-链霉亲和素、配体/受体、酶/底物、核酸/核酸结合蛋白、脂质/脂质结合蛋白、细胞黏着分子配偶体；或彼此具有亲和力的其任何结合配偶体或片段。The present disclosure also includes other conjugates wherein an insulin prodrug analog of the invention is attached to the conjugate, optionally by covalent bonding and optionally by a linker. Linkage can be achieved through covalent chemical bonds; physical forces such as electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions. Various non-covalent conjugation systems can be employed, including biotin-streptavidin, ligand/receptor, enzyme/substrate, nucleic acid/nucleic acid binding protein, lipid/lipid binding protein, cell adhesion molecule partners or any binding partner or fragment thereof that has an affinity for each other.
示例性缀合物包括但不限于异源肽或多肽(包括例如血浆蛋白)、靶向肽、免疫球蛋白或其部分(例如可变区、CDR或Fc区)、诊断标记物(例如放射性同位素、荧光团或酶标记物)、聚合物(包括水溶性聚合物)或其它治疗剂或诊断剂。在一个实施方案中，提供包含本公开内容的胰岛素前药类似物和血浆蛋白的缀合物，其中所述血浆蛋白选自白蛋白、运铁蛋白和血纤蛋白原。在一个实施方案中，缀合物的血浆蛋白部分是白蛋白或运铁蛋白。在一个实施方案中，接头包含长为1-约60个原子、或1-30个原子或更长、2-5个原子、2-10个原子、5-10个原子或10-20个原子的链。在一个实施方案中，链原子全部为碳原子。在一个实施方案中，接头骨架中的链原子选自C、O、N和S。可以根据其预计的溶解度(亲水性)来选择链原子和接头，使得提供更易溶解的缀合物。在一个实施方案中，接头提供受到存在于靶组织或器官或细胞的酶或其它催化剂或水解条件裂解的官能团。在一个实施方案中，接头的长度足够长以减小位阻的可能性。如果接头为共价键或肽基键，且缀合物为多肽，则整个缀合物可为融合蛋白。这类肽基接头可以为任何长度。示例性接头的长度为约1-50个氨基酸、5-50、3-5、5-10、5-15或10-30个氨基酸。或者这类融合蛋白可通过本领域普通技术人员已知的重组遗传工程方法产生。Exemplary conjugates include, but are not limited to, heterologous peptides or polypeptides (including, e.g., plasma proteins), targeting peptides, immunoglobulins or portions thereof (e.g., variable regions, CDRs, or Fc regions), diagnostic markers (e.g., radioisotope , fluorophores or enzyme labels), polymers (including water-soluble polymers), or other therapeutic or diagnostic agents. In one embodiment, there is provided a conjugate comprising an insulin prodrug analog of the present disclosure and a plasma protein, wherein the plasma protein is selected from albumin, transferrin and fibrinogen. In one embodiment, the plasma protein portion of the conjugate is albumin or transferrin. In one embodiment, the linker comprises 1 to about 60 atoms, or 1-30 atoms or longer, 2-5 atoms, 2-10 atoms, 5-10 atoms, or 10-20 atoms in length chain. In one embodiment, the chain atoms are all carbon atoms. In one embodiment, the chain atoms in the backbone of the linker are selected from C, O, N and S. Chain atoms and linkers can be chosen based on their predicted solubility (hydrophilicity) so as to provide a more soluble conjugate. In one embodiment, the linker provides a functional group that is cleaved by enzymes or other catalysts or hydrolytic conditions present in the target tissue or organ or cell. In one embodiment, the length of the linker is sufficiently long to reduce the possibility of steric hindrance. If the linker is a covalent or peptidyl bond, and the conjugate is a polypeptide, the entire conjugate can be a fusion protein. Such peptidyl linkers can be of any length. Exemplary linkers are about 1-50 amino acids, 5-50, 3-5, 5-10, 5-15, or 10-30 amino acids in length. Alternatively, such fusion proteins can be produced by recombinant genetic engineering methods known to those of ordinary skill in the art.
缀合物和融合物Conjugates and Fusions
本公开内容还包括其它缀合物，其中本文公开的胰岛素类似物任选通过共价结合和任选通过接头与缀合物部分连接。可以通过共价化学键；诸如静电、氢、离子、范德瓦尔斯或疏水或亲水相互作用等物理力实现连接。可以采用各种非共价偶联系统，包括生物素-抗生物素蛋白、配体/受体、酶/底物、核酸/核酸结合蛋白、脂质/脂质结合蛋白、细胞黏着分子配偶体；或彼此具有亲和力的其任何结合配偶体或片段。The present disclosure also includes other conjugates wherein an insulin analog disclosed herein is linked to the conjugate moiety, optionally by covalent bonding and optionally by a linker. Linkage can be achieved through covalent chemical bonds; physical forces such as electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions. Various non-covalent conjugation systems can be employed, including biotin-avidin, ligand/receptor, enzyme/substrate, nucleic acid/nucleic acid binding protein, lipid/lipid binding protein, cell adhesion molecule partners ; or any binding partner or fragment thereof that has an affinity for each other.
可以通过使肽的靶定氨基酸残基与有机衍生剂反应将肽与缀合物部分经由直接共价键连接，所述有机衍生剂能够与选定侧链或这些靶定氨基酸的N端或C端残基反应。肽或缀合物上的反应基包括例如醛、氨基、酯、巯基、α-卤代乙酰基、马来酰亚胺基或肼基。衍生剂包括例如马来酰亚胺基苯甲酰基磺基琥珀酰亚胺酯(通过半胱氨酸残基缀合)、N-羟基琥珀酰亚胺(通过赖氨酸残基)、戊二醛、琥珀酸酐或本领域已知的其它衍生剂。或者，缀合物部分可以通过中间载体(例如多糖或多肽载体)与肽间接连接。多糖载体的实例包括氨基葡聚糖。合适的多肽载体的实例包括聚赖氨酸、聚谷氨酸、聚天冬氨酸、其共聚物以及这些氨基酸和其它氨基酸(例如丝氨酸)的混合聚合物，以给所得到的负载载体提供所需要的溶解度性质。The peptide and the conjugate moiety can be linked via direct covalent bonds by reacting targeted amino acid residues of the peptide with an organic derivatizing agent capable of binding to selected side chains or the N-terminal or C-terminal of these targeted amino acids. terminal residue reaction. Reactive groups on the peptide or conjugate include, for example, aldehyde, amino, ester, sulfhydryl, alpha-haloacetyl, maleimido or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), pentadiene Aldehydes, succinic anhydride, or other derivatizing agents known in the art. Alternatively, the conjugate moiety can be indirectly linked to the peptide via an intermediate carrier such as a polysaccharide or polypeptide carrier. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and other amino acids (such as serine) to provide the resulting load carrier with the desired Solubility properties required.
半胱氨酰基残基最常与诸如氯乙酸或氯乙酰胺等α-卤代乙酸酯(和相应的胺)反应，得到羧基甲基或羧基酰氨基甲基衍生物。还通过与溴三氟丙酮、α-溴-β-(5-咪唑基)丙酸、氯乙酰基磷酸(chloroacetyl phosphate)、N-烷基马来酰亚胺、3-硝基-2-吡啶基二硫化合物、甲基2-吡啶基二硫化合物、对氯汞苯甲酸、2-氯汞基-4-硝基苯酚或氯-7-硝基苯并-2--1，3-二唑反应，来使半胱氨酰基残基衍生化。Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Also by bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridine disulfide, methyl 2-pyridyl disulfide, p-chloromercury benzoic acid, 2-chloromercuryl-4-nitrophenol or chloro-7-nitrobenzo-2- -1,3-oxadiazole reaction to derivatize cysteinyl residues.
通过在pH 5.5-7.0下与焦碳酸二乙酯反应使组氨酰残基衍生化，因为该衍生剂对组氨酰基侧链相对具有特异性。对溴代苯甲酰甲基溴也是可用的；该反应优选在0.1M二甲胂酸钠中于pH 6.0进行。Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0, since this derivatizing agent is relatively specific for the histidyl side chain. p-Bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
使赖氨酰残基和氨基端残基与琥珀酸酐或其它羧酸酐反应。与这些衍生剂的衍生化具有逆转赖氨酰残基的电荷的作用。用于使含有α-氨基的残基衍生化的其它合适试剂包括亚氨酸酯例如甲基吡啶亚胺甲酯、磷酸吡哆醛、吡哆醛、氯硼氢化物(chloroborohydride)、三硝基苯磺酸、邻甲基异脲、2，4-戊二酮和转氨酶催化的与乙醛酸的反应。Lysinyl residues and amino terminal residues are reacted with succinic anhydride or other carboxylic acid anhydrides. Derivatization with these derivatizing agents has the effect of reversing the charge of the lysyl residues. Other suitable reagents for derivatizing α-amino containing residues include imidate esters such as picolinimine methyl, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro Reaction with glyoxylate catalyzed by benzenesulfonic acid, o-methylisourea, 2,4-pentanedione and transaminases.
通过与一种或若干种常用试剂(其中有苯甲酰甲醛、2，3-丁二酮、1，2-环己二酮和茚三酮)反应来修饰精氨酰残基。因胍官能团的高pKa所致，精氨酸残基的衍生化需要在碱性条件下进行反应。此外，这些试剂可与赖氨酸的基团以及精氨酸ε-氨基反应。Arginyl residues are modified by reaction with one or several commonly used reagents, among which phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residues requires reaction under basic conditions due to the high pKa of the guanidine functional group. In addition, these reagents can react with groups of lysine as well as with the ε-amino group of arginine.
可对酪氨酰残基进行特异修饰，其中特别关注的是通过与芳族重氮化合物或四硝基甲烷反应将光谱标记引入酪氨酰残基。最常见的是分别使用N-乙酰基咪唑(acetylimidizole)和四硝基甲烷形成O-乙酰基酪氨酰基类和3-硝基衍生物。Specific modifications can be made to tyrosyl residues, of particular interest is the introduction of spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. The most common is the use of N-acetylimidizole and tetranitromethane to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.
通过与诸如1-环己基-3-(2-吗啉基-4-乙基)碳二亚胺或1-乙基-3-(4-氮-4，4-二甲基戊基)碳二亚胺等碳二亚胺(R-N＝C＝N-R′)(其中R和R′为不同的烷基)反应，选择性地修饰羧基侧基(天冬氨酰基或谷氨酰基)。此外，通过与铵离子反应，将天冬氨酰残基和谷氨酰残基转化成天冬酰胺酰残基和谷氨酰胺酰残基。By reacting with such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-nitro -4,4-Dimethylpentyl) carbodiimide (RN=C=NR') (wherein R and R' are different alkyl groups) react to selectively modify the carboxyl side group ( aspartyl or glutamyl). In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
其它修饰包括脯氨酸和赖氨酸的羟基化；丝氨酰残基或苏氨酰残基的羟基的磷酸化；赖氨酸、精氨酸和组氨酸侧链的α-氨基的甲基化(T.E.Creighton，Proteins：Structureand Molecular Properties，W.H.Freeman & Co.，San Francisco，第79-86页(1983))、天冬酰胺或谷氨酰胺的脱酰胺化；N端胺的乙酰化和/或C端羧酸基的酰胺化或酯化。Other modifications include hydroxylation of proline and lysine; phosphorylation of the hydroxyl group of seryl or threonyl residues; methylation of the α-amino groups of lysine, arginine, and histidine side chains Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine; acetylation of N-terminal amines and / or amidation or esterification of the C-terminal carboxylic acid group.
另一种类型的共价修饰包括将糖苷与肽进行化学偶联或酶促偶联。可将糖连接至(a)精氨酸和组氨酸，(b)游离羧基，(c)游离巯基(例如半胱氨酸的巯基)，(d)游离羟基(例如丝氨酸、苏氨酸或羟脯氨酸的羟基)，(e)芳族残基(例如酪氨酸或色氨酸的芳族残基)，或(f)谷氨酰胺的酰胺基。这些方法披露于1987年9月11日公布的WO87/05330和Aplin和Wriston，CRC Crit.Rev.Biochem.，第259-306页(1981)。Another type of covalent modification involves chemical or enzymatic coupling of glycosides to the peptide. Sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as serine, threonine or hydroxyproline), (e) an aromatic residue such as that of tyrosine or tryptophan, or (f) an amide group of glutamine. These methods are disclosed in WO 87/05330 published September 11, 1987 and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
可与本文所述的任何胰岛素类似物连接的示例性缀合物部分包括但不限于异源肽或多肽(包括例如血浆蛋白)、靶向剂、免疫球蛋白或其部分(例如可变区、CDR或Fc区)、诊断标记(例如放射性同位素、荧光团或酶标记)、聚合物(包括水溶性聚合物)或其它治疗剂或诊断剂。在一个实施方案中，提供包含本文公开的胰岛素类似物和血浆蛋白的缀合物，其中血浆蛋白选自白蛋白、运铁蛋白、血纤蛋白原和球蛋白。Exemplary conjugate moieties that can be linked to any of the insulin analogs described herein include, but are not limited to, heterologous peptides or polypeptides (including, for example, plasma proteins), targeting agents, immunoglobulins, or portions thereof (e.g., variable regions, CDR or Fc region), diagnostic markers (such as radioisotopes, fluorophores, or enzyme labels), polymers (including water-soluble polymers), or other therapeutic or diagnostic agents. In one embodiment, there is provided a conjugate comprising an insulin analog disclosed herein and a plasma protein, wherein the plasma protein is selected from the group consisting of albumin, transferrin, fibrinogen and globulin.
在一个实施方案中，接头包含长为1-约60个原子、或1-30原子或更长、2-5个原子、2-10个原子、5-10个原子或10-20个原子的链。在一个实施方案中，链原子全部为碳原子。在一个实施方案中，接头骨架中的链原子选自C、O、N和S。可根据其预期的溶解度(亲水性)来选择链原子和接头，以提供更易溶解的缀合物。在一个实施方案中，接头提供受到存在于靶组织或器官或细胞中的酶或其它催化剂或水解条件裂解的官能团。在一个实施方案中，接头的长度足够长以减小位阻的可能性。如果接头为共价键或肽基键，且缀合物为多肽，则整个缀合物可为融合蛋白。这类肽基接头可以为任何长度。示例性接头的长度为约1-50个氨基酸、5-50、3-5、5-10、5-15或10-30个氨基酸。或者，这类融合蛋白可通过本领域普通技术人员已知的重组遗传工程方法产生。In one embodiment, the linker comprises 1 to about 60 atoms, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in length. chain. In one embodiment, the chain atoms are all carbon atoms. In one embodiment, the chain atoms in the backbone of the linker are selected from C, O, N and S. Chain atoms and linkers can be chosen based on their expected solubility (hydrophilicity) to provide a more soluble conjugate. In one embodiment, the linker provides a functional group that is cleaved by enzymes or other catalysts or hydrolytic conditions present in the target tissue or organ or cell. In one embodiment, the length of the linker is sufficiently long to reduce the possibility of steric hindrance. If the linker is a covalent or peptidyl bond, and the conjugate is a polypeptide, the entire conjugate can be a fusion protein. Such peptidyl linkers can be of any length. Exemplary linkers are about 1-50 amino acids, 5-50, 3-5, 5-10, 5-15, or 10-30 amino acids in length. Alternatively, such fusion proteins can be produced by recombinant genetic engineering methods known to those of ordinary skill in the art.
如上所述，在一个实施方案中，胰岛素类似物是缀合的，例如与免疫球蛋白或其部分(例如可变区、CDR或Fc区)融合。已知的免疫球蛋白(Ig)类型包括IgG、IgA、IgE、IgD或IgM。Fc区是Ig重链的C端区，其负责与具有诸如再循环(其导致半寿期延长)、依赖抗体的细胞毒性(ADCC)和依赖补体的细胞毒性(CDC)等活性的Fc受体结合。As mentioned above, in one embodiment the insulin analog is conjugated, eg fused to an immunoglobulin or part thereof (eg variable region, CDR or Fc region). Known immunoglobulin (Ig) classes include IgG, IgA, IgE, IgD or IgM. The Fc region is the C-terminal region of the Ig heavy chain that is responsible for interacting with Fc receptors with activities such as recycling (which leads to increased half-life), antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) combined.
例如，根据某些定义，人IgG重链Fc区自Cys226延伸到重链的C端。“铰链区”一般自人IgG1的Glu216延伸到Pro230(可通过比对参与半胱氨酸结合的半胱氨酸，来将其它IgG同种型的铰链区与IgG1序列进行比对)。IgG的Fc区包括2个恒定结构域CH2和CH3。人IgG Fc区的CH2结构域通常自氨基酸231延伸至氨基酸341。人IgG Fc区的CH3结构域通常自氨基酸342延伸至氨基酸447。所提及的免疫球蛋白或免疫球蛋白片段或者区段的氨基酸编号都基于Kabat等，1991，Sequences of Proteins of Immunological Interest，U.S.Departmentof Public Health，Bethesda，Md。在一个相关的实施方案中，Fc区可包含免疫球蛋白重链的一个或多个天然或修饰的恒定区(除CH1以外)，例如IgG和IgA的CH2和CH3区，或者IgE的CH3和CH4区。For example, according to certain definitions, the human IgG heavy chain Fc region extends from Cys226 to the C-terminus of the heavy chain. The "hinge region" generally extends from Glu216 to Pro230 of human IgGl (hinge regions of other IgG isotypes can be aligned to the IgGl sequence by aligning the cysteines involved in cysteine binding). The Fc region of IgG includes two constant domains CH2 and CH3. The CH2 domain of the human IgG Fc region generally extends from amino acid 231 to amino acid 341. The CH3 domain of the human IgG Fc region generally extends from amino acid 342 to amino acid 447. Amino acid numbering of references to immunoglobulins or immunoglobulin fragments or segments is based on Kabat et al., 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md. In a related embodiment, the Fc region may comprise one or more native or modified constant regions (other than CH1) of an immunoglobulin heavy chain, such as the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE Area.
合适的缀合物部分包括包含FcRn结合部位的免疫球蛋白序列的部分。FcRn，一种补救受体，负责使免疫球蛋白再循环，并将其送回血液循环中。根据X-射线晶体学，已描述了结合FcRn受体的IgG Fc部分的区域(Burmeister等，1994，Nature 372：379)。Fc与FcRn的主要接触区域接近CH2和CH3结构域的连接点。Fc-FcRn接触处全都在单一Ig重链内。主要接触部位包括CH2结构域的氨基酸残基248、250-257、272、285、288、290-291、308-311和314以及CH3结构域的氨基酸残基385-387、428和433-436。Suitable conjugate moieties include moieties of immunoglobulin sequences comprising an FcRn binding site. FcRn, a salvage receptor, is responsible for recycling immunoglobulins and sending them back into the blood circulation. The region of the Fc portion of IgG that binds the FcRn receptor has been described based on X-ray crystallography (Burmeister et al., 1994, Nature 372:379). The main contact region between Fc and FcRn is close to the junction of CH2 and CH3 domains. The Fc-FcRn contacts are all within a single Ig heavy chain. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain and amino acid residues 385-387, 428 and 433-436 of the CH3 domain.
一些缀合物部分可包括或者可不包括FcγR结合部位。FcγR负责ADCC和CDC。与FcγR进行直接接触的Fc区内的位置的实例为氨基酸234-239(下铰链区)、氨基酸265-269(B/C环)、氨基酸297-299(C′/E环)和氨基酸327-332(F/G)环(Sondermann等，Nature 406：267-273，2000)。IgE的下铰链区还涉及FcRI结合(Henry等，Biochemistry 36，15568-15578，1997)。参与IgA受体结合的残基参见Lewis等(J Immunol.175：6694-701，2005)。参与IgE受体结合的氨基酸残基参见Sayers等(J Biol Chem.279(34)：35320-5，2004)。Some conjugate moieties may or may not include an FcyR binding site. FcγRs are responsible for ADCC and CDC. Examples of positions within the Fc region that make direct contact with an FcγR are amino acids 234-239 (lower hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C'/E loop), and amino acids 327- 332(F/G) ring (Sondermann et al., Nature 406:267-273, 2000). The lower hinge region of IgE is also involved in FcRI binding (Henry et al., Biochemistry 36, 15568-15578, 1997). See Lewis et al. (J Immunol. 175:6694-701, 2005) for residues involved in IgA receptor binding. For amino acid residues involved in IgE receptor binding, see Sayers et al. (J Biol Chem. 279(34):35320-5, 2004).
可对免疫球蛋白的Fc区进行氨基酸修饰。这类变体Fc区在Fc区的CH3结构域(残基342-447)中包含至少一个氨基酸修饰和/或在Fc区的CH2结构域(残基231-341)中包含至少一个氨基酸修饰。被认为赋予对FcRn提高的亲和力的突变包括T256A、T307A、E380A和N434A(Shields等，2001，J.Biol.Chem.276：6591)。其它突变可减少Fc区与FcγRI、FcγRIIA、FcγRIIB和/或FcγRIIIA的结合而又不显著降低对FcRn的亲和力。例如，Fc区297位的Asn用Ala或另一个氨基酸取代去除高度保守的N-糖基化位点，并可导致Fc区的免疫原性降低同时半寿期延长，以及与FcγR的结合减少(Routledge等，1995，Transplantation 60：847；Friend等，1999，Transplantation 68：1632；Shields等，1995，J.Biol.Chem.276：6591)。已进行了减少与FcγR结合的IgG1 233-236位上的氨基酸修饰(Ward和Ghetie 1995，Therapeutic Immunology 2：77和Armour等1999，Eur.J.Immunol.29：2613)。一些示例性的氨基酸取代参见美国专利7,355,008和7,381,408，通过引用以其整体各自结合到本文中。Amino acid modifications can be made to the Fc region of an immunoglobulin. Such variant Fc regions comprise at least one amino acid modification in the CH3 domain of the Fc region (residues 342-447) and/or at least one amino acid modification in the CH2 domain of the Fc region (residues 231-341). Mutations thought to confer increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al., 2001, J. Biol. Chem. 276:6591). Other mutations can reduce Fc region binding to FcyRI, FcyRIIA, FcyRIIB and/or FcyRIIIA without significantly reducing affinity for FcRn. For example, substitution of Asn at position 297 of the Fc region with Ala or another amino acid removes the highly conserved N-glycosylation site and can lead to reduced immunogenicity of the Fc region with increased half-life and reduced binding to FcγRs ( Routledge et al., 1995, Transplantation 60:847; Friend et al., 1999, Transplantation 68:1632; Shields et al., 1995, J. Biol. Chem. 276:6591). Amino acid modifications at positions 233-236 of IgG1 that reduce binding to FcyRs have been made (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutions are found in US Patent Nos. 7,355,008 and 7,381,408, each incorporated herein by reference in its entirety.
酰化和烷基化Acylation and Alkylation
根据一个实施方案，对本文公开的胰岛素类似物进行修饰以包含酰基或烷基。酰化或烷基化可以延长胰岛素类似物在循环中的半寿期。酰化或烷基化可有利地延迟起效和/或延长在胰岛素受体和/或IGF-1受体上的作用持续时间和/或提高对蛋白酶(例如DPP-IV)的抗性和/或提高溶解度。可以在连接亲水部分的同一氨基酸位置上，或在不同的氨基酸位置上对胰岛素类似物进行酰化或烷基化。According to one embodiment, the insulin analogs disclosed herein are modified to contain an acyl or alkyl group. Acylation or alkylation can prolong the half-life of insulin analogues in circulation. Acylation or alkylation can advantageously delay the onset of action and/or prolong the duration of action at the insulin receptor and/or IGF-1 receptor and/or increase resistance to proteases (such as DPP-IV) and/or or increase solubility. The insulin analog can be acylated or alkylated at the same amino acid position where the hydrophilic moiety is attached, or at a different amino acid position.
在一个实施方案中，本发明提供胰岛素类似物，其经修饰以包含与在相当于天然胰岛素A10、B28、B29的位置上，或者在A链或B链的C端或N端上的氨基酸共价连接的酰基或烷基。胰岛素类似物在胰岛素类似物氨基酸与酰基或烷基之间还可包含间隔物。在一个实施方案中，酰基为脂肪酸或胆汁酸或其盐，例如C4-C30脂肪酸、C8-C24脂肪酸、胆酸、C4-C30烷基、C8-C24烷基，或包含胆汁酸的类固醇部分的烷基。间隔物是具有用于连接酰基或烷基的合适反应基的任何部分。在示例性的实施方案中，间隔物包含氨基酸、二肽或三肽，或亲水双官能间隔物。在一个实施方案中，间隔物选自：Trp、Glu、Asp、Cys和包含NH2(CH2CH2O)n(CH2)mCOOH的间隔物，其中m为1-6的任何整数，n为2-12的任何整数。这类酰化或烷基化胰岛素肽还可另包含亲水部分，任选聚乙二醇。前述胰岛素类似物中的任一种可包含两个酰基或两个烷基，或其组合。In one embodiment, the invention provides an insulin analogue modified to contain an amino acid co-dependent with an amino acid at a position corresponding to native insulin A10, B28, B29, or at the C- or N-terminus of the A or B chains. an acyl or alkyl group attached to the valence. The insulin analog may also comprise a spacer between the insulin analog amino acid and the acyl or alkyl group. In one embodiment, the acyl group is a fatty acid or a bile acid or a salt thereof, such as a C4-C30 fatty acid, a C8-C24 fatty acid, a cholic acid, a C4-C30 alkyl, a C8-C24 alkyl, or a steroid moiety comprising a bile acid. alkyl. A spacer is any moiety having a suitable reactive group for attaching an acyl or alkyl group. In exemplary embodiments, the spacer comprises an amino acid, a dipeptide or tripeptide, or a hydrophilic bifunctional spacer. In one embodiment, the spacer is selected from: Trp, Glu, Asp, Cys and a spacer comprisingNH2 (CH2CH2O )n(CH2) mCOOH, wherein m is any integer from 1 to 6, n Any integer from 2-12. Such acylated or alkylated insulin peptides may further comprise a hydrophilic moiety, optionally polyethylene glycol. Any of the aforementioned insulin analogs may contain two acyl groups or two alkyl groups, or a combination thereof.
可以在胰岛素类似物内的任何位置上进行酰化，前提是保持胰岛素类似物胰岛素激动剂活性。酰基可直接与胰岛素类似物的氨基酸共价连接，或通过间隔物间接与胰岛素类似物的氨基酸共价连接，其中所述间隔物位于胰岛素肽的氨基酸与酰基之间。在本发明的一个具体方面，通过对胰岛素肽的氨基酸侧链的胺、羟基或巯基的直接酰化，将胰岛素类似物修饰成包含酰基。在一个实施方案中，胰岛素类似物通过氨基酸的侧链胺、羟基或巯基直接酰化。在一个实施方案中，酰化位于相当于天然胰岛素的A10、B28、B29或者A链或B链的C端或N端。在这点上，酰化胰岛素类似物可包含SEQ ID NO：9和SEQ ID NO：10的氨基酸序列，或其修饰氨基酸序列，该序列包含一个或多个本文所述氨基酸修饰，其中在相当于天然胰岛素的A10、B28、B29的位置上或者A链或B链的C端或N端中的至少一个氨基酸被修饰成包含侧链胺、羟基或巯基的任何氨基酸。在一些具体的实施方案中，通过在相当于天然胰岛素的A10、B28、B29位上的氨基酸的侧链胺、羟基或巯基进行胰岛素肽的直接酰化。根据一个实施方案，二肽元件的氨基酸侧链之一被酰化。Acylation can be performed at any position within the insulin analog provided that insulin agonist activity of the insulin analog is maintained. The acyl group may be covalently linked directly to the amino acid of the insulin analog, or indirectly through a spacer between the amino acid and the acyl group of the insulin peptide. In a specific aspect of the invention, the insulin analogue is modified to contain an acyl group by direct acylation of an amine, hydroxyl or sulfhydryl group of an amino acid side chain of the insulin peptide. In one embodiment, the insulin analog is directly acylated via the side chain amine, hydroxyl or thiol of an amino acid. In one embodiment, the acylation is at the C- or N-terminus corresponding to A10, B28, B29, or the A or B chain of native insulin. In this regard, the acylated insulin analogue may comprise the amino acid sequence of SEQ ID NO: 9 and SEQ ID NO: 10, or a modified amino acid sequence thereof comprising one or more of the amino acid modifications described herein, wherein At least one amino acid at the position of A10, B28, B29 of natural insulin or in the C-terminal or N-terminal of A chain or B chain is modified to any amino acid containing side chain amine, hydroxyl or sulfhydryl. In some specific embodiments, direct acylation of the insulin peptide is carried out via the side chain amine, hydroxyl or sulfhydryl group of amino acids corresponding to positions A10, B28, B29 of native insulin. According to one embodiment, one of the amino acid side chains of the dipeptide element is acylated.
在一个实施方案中，待酰化的氨基酸为下式IV的氨基酸：In one embodiment, the amino acid to be acylated is the amino acid of formula IV below:
其中n＝1-4where n=1-4
[式IV][Formula IV]
在一些示例性的实施方案中，式IV的氨基酸是其中n为4(Lys)或n为3(Orn)的氨基酸。In some exemplary embodiments, the amino acid of Formula IV is the amino acid wherein n is 4 (Lys) or n is 3 (Orn).
在其它实施方案中，包含侧链羟基的氨基酸为下式V的氨基酸：In other embodiments, the amino acid comprising a side chain hydroxyl group is an amino acid of Formula V below:
其中n＝1-4where n=1-4
[式V][Formula V]
在一些示例性的实施方案中，式V氨基酸是其中n为1的氨基酸(Ser)。In some exemplary embodiments, the amino acid of Formula V is the amino acid wherein n is 1 (Ser).
在其它实施方案中，包含侧链巯基的氨基酸为下式VI的氨基酸：In other embodiments, the amino acid comprising a side chain sulfhydryl group is an amino acid of formula VI below:
其中n＝1-4where n=1-4
[式VI][Formula VI]
在一些示例性的实施方案中，式VI氨基酸为其中n为1的氨基酸(Cys)。In some exemplary embodiments, the amino acid of Formula VI is an amino acid wherein n is 1 (Cys).
在一些示例性的实施方案中，通过对间隔物的胺、羟基或巯基的酰化来修饰胰岛素类似物以包含酰基，所述间隔物与在A10、B28或B29位(按照野生型胰岛素的氨基酸编号)上的氨基酸的侧链连接。间隔物与之连接的氨基酸可以是包含允许与间隔物连接的部分的任何氨基酸。例如包含侧链NH2、-OH或-COOH的氨基酸(例如Lys、Orn、Ser、Asp或Glu)是适宜的。在一个实施方案中，间隔物是包含侧链胺、羟基或巯基的氨基酸，或者是包含含有侧链胺、羟基或巯基的氨基酸的二肽或三肽。In some exemplary embodiments, the insulin analog is modified to include an acyl group by acylation of the amine, hydroxyl, or sulfhydryl group of a spacer that is compatible with amino acids at positions A10, B28, or B29 (according to wild-type insulin). The side chain linkages of the amino acids on the number). The amino acid to which the spacer is attached may be any amino acid comprising a moiety which allows attachment of the spacer. For example amino acids comprising side chainsNH2 , -OH or -COOH (eg Lys, Orn, Ser, Asp or Glu) are suitable. In one embodiment, the spacer is an amino acid comprising a side chain amine, hydroxyl or sulfhydryl, or a dipeptide or tripeptide comprising an amino acid comprising a side chain amine, hydroxyl or sulfhydryl.
当通过间隔物的胺基团进行酰化时，酰化可通过氨基酸的α胺或侧链胺进行。在其中α胺被酰化的情况下，间隔物氨基酸可以是任何氨基酸。例如，间隔物氨基酸可以是疏水氨基酸，例如Gly、Ala、Val、Leu、Ile、Trp、Met、Phe、Tyr。或者，间隔物氨基酸可以是酸性残基，例如Asp和Glu。在其中间隔物氨基酸的侧链胺被酰化的情况下，间隔物氨基酸是包含侧链胺的氨基酸，例如式I氨基酸(例如Lys或Orn)。在这种情况下，间隔物氨基酸的α胺和侧链胺两个都可能被酰化，使得胰岛素肽是二酰化的。本公开内容还考虑二酰化胰岛素类似物。When the acylation is through the amine group of the spacer, the acylation can be through the alpha amine or the side chain amine of the amino acid. In cases where the alpha amine is acylated, the spacer amino acid can be any amino acid. For example, the spacer amino acid can be a hydrophobic amino acid such as Gly, Ala, Val, Leu, Ile, Trp, Met, Phe, Tyr. Alternatively, the spacer amino acids may be acidic residues such as Asp and Glu. In cases where the side chain amine of the spacer amino acid is acylated, the spacer amino acid is an amino acid comprising a side chain amine, eg an amino acid of formula I (eg Lys or Orn). In this case, both the alpha amine and the side chain amine of the spacer amino acid may be acylated such that the insulin peptide is diacylated. The present disclosure also contemplates diacylated insulin analogs.
当酰化通过间隔物的羟基进行时，所述氨基酸或者二肽或三肽的氨基酸之一可以为式II氨基酸。在一个具体的示例性实施方案中，该氨基酸为Ser。The amino acid or one of the amino acids of the dipeptide or tripeptide may be an amino acid of formula II when the acylation is via the hydroxyl group of the spacer. In a specific exemplary embodiment, the amino acid is Ser.
当酰化通过间隔物的巯基进行时，所述氨基酸或者二肽或三肽的氨基酸之一可为式III氨基酸。在一个具体的示例性实施方案中，该氨基酸为Cys。The amino acid or one of the amino acids of the dipeptide or tripeptide may be an amino acid of formula III when the acylation is via the sulfhydryl group of the spacer. In a specific exemplary embodiment, the amino acid is Cys.
在一个实施方案中，间隔物包含亲水双官能间隔物。在一个具体的实施方案中，间隔物包含氨基聚(烷氧基)羧酸。在这点上，间隔物可包含例如NH2(CH2CH2O)n(CH2)mCOOH，其中m为1-6的任何整数，n为2-12的任何整数，例如8-氨基-3，6-二氧杂辛酸，可购自PeptidesInternational，Inc.(Louisville，KY)。In one embodiment, the spacer comprises a hydrophilic bifunctional spacer. In a specific embodiment, the spacer comprises an aminopoly(alkoxy)carboxylic acid. In this regard, the spacer may comprise, for example,NH2 (CH2CH2O )n (CH2 )mCOOH, wherem is any integer from 1 to 6 and n is any integer from2 to 12, such as 8-amino - 3,6-dioxahanoic acid, commercially available from Peptides International, Inc. (Louisville, KY).
通过胺、羟基和巯基进行肽酰化的合适方法是本领域已知的。参见例如Miller，Biochem Biophys Res Commun 218：377-382(1996)；Shimohigashi和Stammer，Int J PeptProtein Res 19：54-62(1982)；Previero等，Biochim Biophys Acta 263：7-13(1972)(用于通过羟基进行酰化的方法)；San和Silvius，J Pept Res 66：169-180(2005)(用于通过巯基进行酰化的方法)；Bioconjugate Chem.“Chemical Modifications of Proteins：History and Applications”第1，2-12页(1990)；Hashimoto等，Pharmacuetical Res.“Synthesis of Palmitoyl Derivatives of Insulin and their Biological Activity”第6卷，第2期第171-176页(1989)。Suitable methods for peptide acylation via amines, hydroxyls and sulfhydryls are known in the art. See, e.g., Miller, Biochem Biophys Res Commun 218:377-382 (1996); Shimohigashi and Stammer, Int J Pept Protein Res 19:54-62 (1982); Previero et al., Biochim Biophys Acta 263:7-13 (1972) (with for acylation via hydroxyl groups); San and Silvius, J Pept Res 66:169-180 (2005) (for acylation via sulfhydryl groups); Bioconjugate Chem. "Chemical Modifications of Proteins: History and Applications" pp. 1, 2-12 (1990); Hashimoto et al., Pharmaceutical Res. "Synthesis of Palmitoyl Derivatives of Insulin and their Biological Activity" Vol. 6, No. 2, pp. 171-176 (1989).
酰化胰岛素肽的酰基可为任何大小，例如任何长度的碳链，并且可以是直链或支链的。在本发明的一些具体实施方案中，酰基为C4-C30脂肪酸。例如，酰基可为以下脂肪酸中的任一种：C4脂肪酸、C6脂肪酸、C8脂肪酸、C10脂肪酸、C12脂肪酸、C14脂肪酸、C16脂肪酸、C18脂肪酸、C20脂肪酸、C22脂肪酸、C24脂肪酸、C26脂肪酸、C28脂肪酸或C30脂肪酸。在一个实施方案中，酰基为C8-C20脂肪酸，例如C14脂肪酸或C16脂肪酸。The acyl group of the acylated insulin peptide can be of any size, eg, carbon chain of any length, and can be linear or branched. In some embodiments of the invention, the acyl group is a C4-C30 fatty acid. For example, the acyl group can be any of the following fatty acids: C4 fatty acid, C6 fatty acid, C8 fatty acid, C10 fatty acid, C12 fatty acid, C14 fatty acid, C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fatty acid, C28 fatty acid fatty acids or C30 fatty acids. In one embodiment, the acyl group is a C8-C20 fatty acid, such as a C14 fatty acid or a C16 fatty acid.
在一个备选实施方案中，酰基为胆汁酸。胆汁酸可以是任何合适的胆汁酸，包括但不限于胆酸、鹅脱氧胆酸、脱氧胆酸、石胆酸、牛磺胆酸、甘氨胆酸和胆固醇酸(cholesterolacid)。In an alternative embodiment, the acyl group is a bile acid. The bile acid may be any suitable bile acid including, but not limited to, cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterololacid.
在一个具体的实施方案中，胰岛素类似物包含胆固醇酸，其通过烷基化脱氨基Cys间隔物(即烷基化3-巯基丙酸间隔物)与胰岛素类似物的Lys残基连接。烷基化脱氨基Cys间隔物可以为例如包含十二乙二醇部分的脱氨基-Cys间隔物。在一个实施方案中，胰岛素类似物包含以下结构：In a specific embodiment, the insulin analog comprises cholesteric acid linked to a Lys residue of the insulin analog via an alkylated deamino Cys spacer (ie, an alkylated 3-mercaptopropionic acid spacer). The alkylated deamino Cys spacer can be, for example, a deamino-Cys spacer comprising a dodecaethylene glycol moiety. In one embodiment, the insulin analog comprises the following structure:
本文所述的酰化胰岛素类似物可进一步修饰以包含亲水部分。在一些具体的实施方案中，亲水部分可包含聚乙二醇(PEG)链。亲水部分的掺入可通过任何合适的方法完成，例如本文所述的任何方法。The acylated insulin analogs described herein can be further modified to include hydrophilic moieties. In some specific embodiments, the hydrophilic portion may comprise polyethylene glycol (PEG) chains. Incorporation of hydrophilic moieties can be accomplished by any suitable method, such as any described herein.
或者，酰化胰岛素肽可包含间隔物，其中对间隔物进行酰化并且修饰以包含亲水部分。合适间隔物的非限制性实例包括包含选自Cys、Lys、Orn、高Cys和Ac-Phe中的一个或多个氨基酸的间隔物。Alternatively, the acylated insulin peptide may comprise a spacer, wherein the spacer is acylated and modified to include a hydrophilic moiety. Non-limiting examples of suitable spacers include spacers comprising one or more amino acids selected from Cys, Lys, Orn, homoCys, and Ac-Phe.
根据一个实施方案，对胰岛素类似物进行修饰以包含通过酯、醚、硫醚、酰胺或烷基胺键与胰岛素类似物连接的烷基，用于延长循环中的半寿期和/或延迟起效和/或延长作用持续时间和/或提高对蛋白酶(例如DPP-IV)的抗性。According to one embodiment, the insulin analogue is modified to include an alkyl group attached to the insulin analogue via an ester, ether, thioether, amide or alkylamine linkage for increasing half-life in circulation and/or delaying onset effect and/or prolong the duration of action and/or increase resistance to proteases (eg DPP-IV).
烷基化胰岛素肽的烷基可以是任何大小，例如任何长度的碳链，且可以是直链或支链的。在本发明的一个实施方案中，烷基为C1-C30烷基。例如，烷基可以是以下烷基中的任一种：C1烷基、C2烷基、C3烷基、C4烷基、C6烷基、C8烷基、C10烷基、C12烷基、C14烷基、C16烷基、C18烷基、C20烷基、C22烷基、C24烷基、C26烷基、C28烷基或C30烷基。在一个实施方案中，烷基为C8-C20烷基，例如C14烷基或C16烷基。The alkyl group of an alkylated insulin peptide may be of any size, eg carbon chain of any length, and may be linear or branched. In one embodiment of the present invention, the alkyl group is a C1-C30 alkyl group. For example, the alkyl group can be any of the following: C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl , C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28 alkyl or C30 alkyl. In one embodiment, the alkyl group is a C8-C20 alkyl group, such as a C14 alkyl group or a C16 alkyl group.
在一些具体的实施方案中，烷基包含胆汁酸的类固醇部分，所述胆汁酸例如为胆酸、鹅脱氧胆酸、脱氧胆酸、石胆酸、牛磺胆酸、甘氨胆酸和胆固醇酸。In some specific embodiments, the alkyl group comprises a steroidal moiety of a bile acid such as cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.
根据一个实施方案，提供包含本文公开的任何新的胰岛素前药类似物和药学上可接受的稀释剂、载体或赋形剂的药物组合物，优选所述胰岛素前药类似物的纯度水平为至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％。这类组合物可含有本文公开的A19胰岛素类似物，浓度为至少0.5mg/ml、1mg/ml、2mg/ml、3mg/ml、4mg/ml、5mg/ml、6mg/ml、7mg/ml、8mg/ml、9mg/ml、10mg/ml、11mg/ml、12mg/ml、13mg/ml、14mg/ml、15mg/ml、16mg/ml、17mg/ml、18mg/ml、19mg/ml、20mg/ml、21mg/ml、22mg/ml、23mg/ml、24mg/ml、25mg/ml或更高。在一个实施方案中，药物组合物包含经过灭菌的水溶液剂，任选装入各种包装容器中保存。在其它实施方案中，药物组合物包含冻干散剂。药物组合物还可作为试剂盒的部分包装，所述试剂盒包括用于将所述组合物给予患者的一次性装置。容器或试剂盒可标上在环境室温下保存或在冷冻温度下保存的标签。According to one embodiment, there is provided a pharmaceutical composition comprising any novel insulin prodrug analog disclosed herein and a pharmaceutically acceptable diluent, carrier or excipient, preferably said insulin prodrug analog has a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Such compositions may contain an A19 insulin analog disclosed herein at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, 20mg/ml ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml or higher. In one embodiment, the pharmaceutical composition comprises a sterilized aqueous solution, optionally stored in various packaging containers. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may also be packaged as part of a kit comprising a disposable device for administering the composition to a patient. The container or kit can be labeled for storage at ambient room temperature or storage at refrigerated temperature.
在一个实施方案中，提供包含第一和第二胰岛素前药类似物的混合物的组合物，其中第一和第二胰岛素前药类似物根据前药元件结构而彼此不同。更具体地讲，第一胰岛素前药类似物可包含半寿期与第二胰岛素前药类似物的二肽前药元件的半寿期基本不同的二肽前药元件。因此，选择二肽元件上取代基的不同组合可供制备以下组合物，其包含在所需时限和特定的时间间隔内以受控方式激活的胰岛素前药类似物的混合物。例如，可将组合物制成餐时释放活性胰岛素，接着在夜间根据活化时间释放出适当剂量的活化胰岛素。在另一个实施方案中，药物组合物包含本文公开的胰岛素前药类似物和天然胰岛素或胰岛素的已知生物活性衍生物的混合物。In one embodiment, there is provided a composition comprising a mixture of first and second insulin prodrug analogs, wherein the first and second insulin prodrug analogs differ from each other by the structure of the prodrug element. More specifically, the first insulin prodrug analog may comprise a dipeptide prodrug element having a half-life substantially different from the half-life of the dipeptide prodrug element of the second insulin prodrug analog. Thus, selection of different combinations of substituents on the dipeptide element allows for the preparation of compositions comprising a mixture of insulin prodrug analogs activated in a controlled manner over a desired time frame and at specific time intervals. For example, the composition can be formulated to release active insulin during meals followed by an appropriate dose of activated insulin at night depending on the time of activation. In another embodiment, a pharmaceutical composition comprises a mixture of an insulin prodrug analog disclosed herein and native insulin or a known biologically active derivative of insulin.
所公开的胰岛素前药类似物被认为适于之前已对胰岛素肽所述的任何用途。因此，本文所述的胰岛素前药类似物可用于治疗高血糖症，或治疗由高血糖水平所致的其它代谢病。因此，本发明包括包含本公开内容的胰岛素前药类似物和药学上可接受的载体的药物组合物，用于治疗患有高血糖水平的患者。根据一个实施方案，待使用本文公开的胰岛素前药类似物治疗的患者为驯养动物，而在另一个实施方案中，待治疗的患者为人。The disclosed insulin prodrug analogs are considered suitable for any of the uses previously described for insulin peptides. Accordingly, the insulin prodrug analogs described herein are useful in the treatment of hyperglycemia, or in the treatment of other metabolic diseases caused by high blood glucose levels. Accordingly, the present invention includes pharmaceutical compositions comprising an insulin prodrug analog of the present disclosure and a pharmaceutically acceptable carrier for use in treating a patient suffering from high blood glucose levels. According to one embodiment, the patient to be treated with the insulin prodrug analogs disclosed herein is a domesticated animal, while in another embodiment the patient to be treated is a human.
按照本公开内容治疗高血糖症的一种方法包括采用任何标准给药途径将本发明公开的胰岛素前药类似物给予患者的步骤，所述给药途径包括胃肠外，例如静脉内、腹膜内、皮下或肌内、鞘内、经皮、直肠、口服、经鼻或通过吸入。在一个实施方案中，皮下或肌内给予组合物。在一个实施方案中，胃肠外给予组合物，且将胰岛素前药类似物组合物预先包装在注射器中。One method of treating hyperglycemia according to the present disclosure comprises the step of administering to a patient an insulin prodrug analog disclosed herein by any standard route of administration, including parenteral, e.g., intravenous, intraperitoneal , subcutaneously or intramuscularly, intrathecally, transdermally, rectally, orally, nasally or by inhalation. In one embodiment, the composition is administered subcutaneously or intramuscularly. In one embodiment, the composition is administered parenterally, and the insulin prodrug analog composition is prepackaged in a syringe.
本发明的胰岛素前药类似物可单独或与其它抗糖尿病药组合给予。本领域已知的或正在研究中的抗糖尿病药包括天然胰岛素；天然胰高血糖素及其功能衍生物；磺脲类，例如甲苯磺丁脲(甲糖宁(Orinase))、醋酸己脲(戴美乐(Dymelor))、妥拉磺脲(妥拉磺脲片剂(Tolinase))、氯磺丙脲(特泌胰(Diabinese))、格列吡嗪((利糖妥片(Glucotrol))、格列本脲(达安宁(Diabeta)、优降糖(Micronase)、达安辽(Glynase))、格列美脲(亚莫利(Amaryl))或格列齐特(达美康(Diamicron))；氯茴苯酸类，例如瑞格列奈(repaglinide)(Prandin)或那格列奈(nateglinide)(Starlix)；双胍类，例如二甲双胍(格华止(Glucophage))或苯乙双胍；噻唑烷二酮类，例如罗格列酮(文迪雅(Avandia))、吡格列酮(pioglitazone)(Actos)或曲格列酮(瑞如林(Rezulin))或其它PPARγ抑制剂；抑制碳水化合物消化的α葡糖苷酶抑制剂，例如米格列醇(德赛天(Glyset))、阿卡波糖(阿卡糖(Precose)/拜糖平(Glucobay))；艾塞那肽(百泌达(Byetta))或普兰林肽；二肽基肽酶-4(DPP-4)抑制剂，例如维格列汀或西格列汀；SGLT(钠依赖性葡萄糖转运蛋白1)抑制剂；或FBP酶(果糖1，6-二磷酸酶)抑制剂。The insulin prodrug analogs of the invention may be administered alone or in combination with other antidiabetic agents. Antidiabetic agents known in the art or under investigation include natural insulin; natural glucagon and its functional derivatives; sulfonylureas, such as tolbutamide (Orinase), hexylurea acetate ( Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol ), glibenclamide (Diabeta, Micronase, Glynase), glimepiride (Amaryl) or gliclazide (Diabeta ( Diamicron)); meglitinides such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin ; thiazolidinediones, such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin) or other PPAR gamma inhibitors; inhibiting carbohydrates Digestive alpha glucosidase inhibitors such as Miglitol (Glyset), Acarbose (Precose/Glucobay); Exenatide (Bysec Byetta) or pramlintide; dipeptidyl peptidase-4 (DPP-4) inhibitors such as vildagliptin or sitagliptin; SGLT (sodium-dependent glucose transporter 1) inhibitors; or FBPase (fructose 1,6-bisphosphatase) inhibitors.
可采用标准的药学上可接受的载体和本领域技术人员已知的给药途径，配制包含本文公开的胰岛素前药类似物的药物组合物并给予患者。因此，本公开内容还包括包含一种或多种本文公开的胰岛素前药类似物或其药学上可接受的盐以及药学上可接受的载体的药物组合物。在一个实施方案中，药物组合物包含在磷酸盐缓冲系统中的pH为约4.0-约7.0、浓度为1mg/ml的胰岛素前药类似物。药物组合物可包含作为唯一的药学活性组分的胰岛素前药类似物，或者胰岛素前药类似物可与一种或多种其它活性剂组合。根据一个实施方案，提供包含本文公开的胰岛素前药类似物之一和药学上可接受的稀释剂、载体或赋形剂的药物组合物，所述胰岛素前药类似物优选为无菌的，且优选纯度水平为至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％。这类组合物可含有胰岛素前药类似物，其中所得活性肽存在的浓度为至少0.5mg/ml、1mg/ml、2mg/ml、3mg/ml、4mg/ml、5mg/ml、6mg/ml、7mg/ml、8mg/ml、9mg/ml、10mg/ml、11mg/ml、12mg/ml、13mg/ml、14mg/ml、15mg/ml、16mg/ml、17mg/ml、18mg/ml、19mg/ml、20mg/ml、21mg/ml、22mg/ml、23mg/ml、24mg/ml、25mg/ml或更高。在一个实施方案中，药物组合物包含经过灭菌的水溶液剂，任选保存在各种容器内。根据一个实施方案，可使用本发明的化合物制备易于注射的预制溶液剂。在其它实施方案中，药物组合物包含冻干散剂。药物组合物还可作为试剂盒的部分包装，所述试剂盒包括用于将组合物给予患者的一次性装置。容器或试剂盒可标上在环境室温下保存或在冷冻温度下保存的标签。Pharmaceutical compositions comprising the insulin prodrug analogs disclosed herein can be formulated and administered to patients using standard pharmaceutically acceptable carriers and routes of administration known to those skilled in the art. Accordingly, the present disclosure also includes pharmaceutical compositions comprising one or more insulin prodrug analogs disclosed herein, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises an insulin prodrug analog at a concentration of 1 mg/ml in a phosphate buffered saline system at a pH of about 4.0 to about 7.0. The pharmaceutical composition may comprise the insulin prodrug analog as the only pharmaceutically active ingredient, or the insulin prodrug analog may be combined with one or more other active agents. According to one embodiment, there is provided a pharmaceutical composition comprising one of the insulin prodrug analogs disclosed herein, preferably sterile, and a pharmaceutically acceptable diluent, carrier or excipient, and Preferred levels of purity are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Such compositions may contain insulin prodrug analogs wherein the resulting active peptide is present at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml ml, 20mg/ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml or higher. In one embodiment, the pharmaceutical compositions comprise sterile aqueous solutions, optionally kept in various containers. According to one embodiment, the compounds of the invention may be used in the preparation of ready-to-inject preformed solutions. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may also be packaged as part of a kit comprising a disposable device for administering the composition to a patient. The container or kit can be labeled for storage at ambient room temperature or storage at refrigerated temperature.
本文描述的所有治疗方法、药物组合物、试剂盒和其它类似实施方案均考虑胰岛素前药类似物包括其所有的药学上可接受的盐。All methods of treatment, pharmaceutical compositions, kits and other similar embodiments described herein contemplate insulin prodrug analogs including all pharmaceutically acceptable salts thereof.
在一个实施方案中，提供具有将胰岛素前药类似物组合物给予患者的装置的试剂盒。试剂盒还可包括各种容器，例如小瓶、管、瓶等。优选试剂盒还包括使用说明书。根据一个实施方案，试剂盒的装置为气雾剂分配装置，其中组合物预包装在气雾剂装置内。在另一个实施方案中，试剂盒包含注射器和针，而在一个实施方案中，胰岛素类似物组合物预包装在注射器中。In one embodiment, a kit having means for administering an insulin prodrug analog composition to a patient is provided. Kits can also include various containers, such as vials, tubes, bottles, and the like. Preferably, the kit also includes instructions for use. According to one embodiment, the device of the kit is an aerosol dispensing device, wherein the composition is prepackaged within the aerosol device. In another embodiment, the kit comprises a syringe and a needle, and in one embodiment, the insulin analog composition is prepackaged in a syringe.
可通过标准合成方法、重组DNA技术或制备肽和融合蛋白的任何其它方法制备本发明的化合物。虽然通过标准重组DNA技术无法表达某些非天然氨基酸，但其制备法是本领域已知的。除标准肽化学反应以外，适用时可通过标准有机化学反应合成包含非肽部分的本发明化合物。The compounds of the invention can be prepared by standard synthetic methods, recombinant DNA techniques, or any other method for preparing peptides and fusion proteins. Although some unnatural amino acids cannot be expressed by standard recombinant DNA techniques, methods for their preparation are known in the art. Compounds of the invention comprising non-peptide moieties may be synthesized by standard organic chemistry reactions, where applicable, in addition to standard peptide chemistry.
实施例1Example 1
胰岛素A链和B链的合成Synthesis of insulin A chain and B chain
采用Boc化学法，在4-甲基二苯甲基胺(MBHA)树脂或4-羟基甲基-苯基乙酰氨基甲基(PAM)树脂上合成胰岛素A链和B链。使用95∶5HF/对甲酚于0℃达1小时，使肽从树脂上裂解。在除去HF且醚沉淀后，将肽溶于50％乙酸水溶液，然后冻干。或者，用Fmoc化学法合成肽。使用三氟乙酸(TFA)/三异丙基甲硅烷(TIS)/H2O(95∶2.5∶2.5)于室温达2小时，使肽从树脂上裂解。通过加入过量的乙醚使肽沉淀，并将沉淀溶于酸性缓冲水溶液。用RP-HPLC监测肽的质量，并通过质谱法(ESI或MALDI)证实。Insulin A chain and B chain were synthesized on 4-methylbenzhydrylamine (MBHA) resin or 4-hydroxymethyl-phenylacetamidomethyl (PAM) resin by Boc chemical method. The peptide was cleaved from the resin using 95:5 HF/p-cresol at 0°C for 1 hour. After removal of HF and ether precipitation, the peptide was dissolved in 50% aqueous acetic acid and lyophilized. Alternatively, peptides were synthesized using Fmoc chemistry. Peptides were cleaved from the resin using trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (95:2.5:2.5) for 2 hours at room temperature. The peptide was precipitated by adding excess diethyl ether, and the precipitate was dissolved in aqueous acidic buffer. The mass of the peptides was monitored by RP-HPLC and confirmed by mass spectrometry (ESI or MALDI).
合成在氨基酸7上具有一个游离半胱氨酸且所有其它半胱氨酸作为乙酰氨基甲基A-(SH)7(Acm)6，11，20保护起来的胰岛素A链。合成在7位上具有一个游离半胱氨酸且其它半胱氨酸作为乙酰氨基甲基B-(SH)7(Acm)19保护起来的胰岛素B链。粗制肽通过常规RP-HPLC纯化。Insulin A chains were synthesized with one free cysteine at amino acid 7 and all other cysteines protected as acetamidomethyl A-(SH)7 (Acm)6,11,20 . The insulin B chain was synthesized with one free cysteine at position 7 and the other cysteine protected as acetamidomethyl B-(SH)7 (Acm)19 . Crude peptides were purified by conventional RP-HPLC.
按照图1所示通用程序，将合成的A链和B链通过其天然二硫键连接彼此连接起来。在室温下，通过溶于DMF或DMSO，并按1∶1摩尔比率与2，2’-联硫基双(5-硝基吡啶)(Npys)反应，使相应的B链活化成Cys7-Npys衍生物。活化用RP-HPLC监测，产物用ESI-MS证实。Following the general procedure shown in Figure 1, the synthetic A and B strands were linked to each other via their natural disulfide linkages. Activate the corresponding B chain to Cys by dissolving in DMF or DMSO and reacting with 2,2'-dithiobis(5 -nitropyridine) (Npys) in a 1:1 molar ratio at room temperature Npys derivatives. Activation was monitored by RP-HPLC and product was confirmed by ESI-MS.
通过以1∶1摩尔比率，总的肽浓度为10mg/ml，使相应的A-(SH)7(Acm)6，11，20和B-(Npys)7(Acm)19溶解，来形成第一个B7-A7二硫键。当链结合反应完成时，将混合物稀释至50％乙酸水溶液的浓度。通过加入碘，同时形成最后的两个二硫键。将40倍摩尔过量的碘加入溶液中，将混合物在室温下再搅拌1小时。加入抗坏血酸水溶液终止反应。混合物用RP-HPLC纯化，最终的化合物用MALDI-MS证实。如图2和表1数据所示，对于胰岛素受体结合，按照该方法制备的合成胰岛素与纯化的胰岛素不相上下。FormationofNo._ A B7-A7 disulfide bond. When the chain conjugation reaction was complete, the mixture was diluted to a concentration of 50% aqueous acetic acid. By adding iodine, the final two disulfide bonds are formed simultaneously. A 40-fold molar excess of iodine was added to the solution, and the mixture was stirred at room temperature for an additional 1 hour. Aqueous ascorbic acid was added to terminate the reaction. The mixture was purified by RP-HPLC and the final compound was confirmed by MALDI-MS. As shown in Figure 2 and the data in Table 1, synthetic insulin prepared according to this method was comparable to purified insulin for insulin receptor binding.
还可以采用允许将非编码氨基酸掺入蛋白质中的系统，体内合成包含修饰氨基酸(例如A19位上的4-氨基苯丙氨酸)的胰岛素肽，所述系统包括例如美国专利号7,045,337和7,083,970教导的系统。Insulin peptides comprising modified amino acids, such as 4-aminophenylalanine at position A19, can also be synthesized in vivo using systems that allow the incorporation of non-coding amino acids into proteins, including, for example, the teachings of U.S. Patent Nos. 7,045,337 and 7,083,970 system.
表1：合成胰岛素相对于天然胰岛素的活性Table 1: Activity of synthetic insulin relative to natural insulin
实施例2Example 2
通过还原性烷基化进行的胺基(N端和赖氨酸)的聚乙二醇化PEGylation of amine groups (N-terminal and lysine) by reductive alkylation
a.合成a. Synthesis
以1∶2∶30摩尔比率，将胰岛素(或胰岛素类似物)、mPEG20k-醛(Aldyhyde)和NaBH3CN溶于乙酸缓冲液(pH 4.1-4.4)。反应溶液由0.1N NaCl、0.2N乙酸和0.1N Na2CO3组成。胰岛素肽浓度约为0.5mg/ml。反应在室温下在6小时内进行。反应程度用RP-HPLC监测，反应物的收率约为50％。Insulin (or insulin analog), mPEG20k-aldehyde (Aldyhyde) andNaBH3CN were dissolved in acetate buffer (pH 4.1-4.4) at a molar ratio of 1:2:30. The reaction solution consisted of 0.1N NaCl, 0.2N aceticacid and 0.1NNa2CO3 . The insulin peptide concentration was approximately 0.5 mg/ml. The reaction proceeded at room temperature over 6 hours. The degree of reaction was monitored by RP-HPLC, and the yield of reactant was about 50%.
b.纯化b.Purification
反应混合物用0.1％TFA稀释2-5倍后，上样至制备型RP-HPLC柱中。HPLC条件：C4柱；流速10ml/分钟；A缓冲液：含10％ACN和0.1％TFA的水溶液；B缓冲液：含0.1％TFA的ACN溶液；线性梯度：B％0-40％(0-80分钟)；将PEG-胰岛素或类似物在约35％缓冲液B中洗脱。所需化合物用MALDI-TOF证实，接着通过亚硫酸盐解(sulftolysis)或胰蛋白酶降解进行化学修饰。The reaction mixture was diluted 2-5 times with 0.1% TFA, and loaded onto a preparative RP-HPLC column. HPLC condition: C4 column; Flow rate 10ml/min; A buffer solution: the aqueous solution containing 10% ACN and 0.1% TFA; B buffer solution: the ACN solution containing 0.1% TFA; Linear gradient: B% 0-40% (0- 80 minutes); PEG-insulin or analog was eluted in about 35% buffer B. Desired compounds were confirmed by MALDI-TOF, followed by chemical modification by sulftolysis or trypsin degradation.
通过N-羟基琥珀酰亚胺酰化进行的胺基(N端和赖氨酸)的聚乙二醇化PEGylation of amine groups (N-terminal and lysine) by N-hydroxysuccinimide acylation
a.合成a. Synthesis
将胰岛素(或胰岛素类似物)与mPEG20k-NHS以1∶1的摩尔比率一起溶于0.1N N-二(羟乙基)甘氨酸缓冲液(pH 8.0)。胰岛素肽浓度约为0.5mg/ml。反应进程用HPLC监测。在室温下2小时后，反应物的收率约为90％。Insulin (or insulin analog) and mPEG20k-NHS were dissolved together in 0.1N N-bis(hydroxyethyl)glycine buffer (pH 8.0) at a molar ratio of 1:1. The insulin peptide concentration was approximately 0.5 mg/ml. The progress of the reaction was monitored by HPLC. After 2 hours at room temperature, the yield of the reaction was about 90%.
b.纯化b.Purification
将反应混合物稀释2-5倍后，加样至RP-HPLC。HPLC条件：C4柱；流速10ml/分钟；A缓冲液：含10％ACN和0.1％TFA的水溶液；B缓冲液：含0.1％TFA的ACN溶液；线性梯度：B％0-40％(0-80分钟)；在约35％B时收集PEG-胰岛素或类似物。所需化合物通过MAIDI-TOF证实，接着通过亚硫酸盐解或胰蛋白酶降解进行化学修饰。After diluting the reaction mixture 2-5 times, it was added to RP-HPLC. HPLC condition: C4 column; Flow rate 10ml/min; A buffer solution: the aqueous solution containing 10% ACN and 0.1% TFA; B buffer solution: the ACN solution containing 0.1% TFA; Linear gradient: B% 0-40% (0- 80 minutes); PEG-insulin or analog was collected at about 35% B. Desired compounds were confirmed by MAIDI-TOF, followed by chemical modification by sulfite hydrolysis or trypsin degradation.
苯丙氨酸芳环上乙酰基的还原性胺化聚乙二醇化Reductive Amination Pegylation of Acetyl Groups on the Aryl Ring of Phenylalanine
a.合成a. Synthesis
将胰岛素(或胰岛素类似物)、mPEG20k-酰肼和NaBH3CN按1∶2∶20的摩尔比率溶于乙酸缓冲液(pH 4.1-4.4)。反应溶液由0.1N NaCl、0.2N乙酸和0.1N Na2CO3组成。在室温下经过24小时，胰岛素或胰岛素类似物的浓度约为0.5mg/ml。反应进程用HPLC监测。反应物的转化约为50％。(通过HPLC计算)Insulin (or insulin analog), mPEG20k-hydrazide and NaBH3 CN were dissolved in acetate buffer (pH 4.1-4.4) at a molar ratio of 1:2:20. The reaction solution consisted of 0.1N NaCl, 0.2N aceticacid and 0.1NNa2CO3 . Over 24 hours at room temperature, the concentration of insulin or insulin analog is about 0.5 mg/ml. The progress of the reaction was monitored by HPLC. The conversion of reactants was about 50%. (calculated by HPLC)
b.纯化b.Purification
反应混合物稀释2-5倍后，加样至RP-HPLC。HPLC条件：C4柱；流速10ml/分钟；A缓冲液：含10％ACN和0.1％TFA的水溶液；B缓冲液：含0.1％TFA的ACN溶液；线性梯度：B％0-40％(0-80分钟)；在约35％B时收集PEG-胰岛素或PEG-胰岛素类似物。所需化合物通过MAIDI-TOF证实，接着通过亚硫酸盐解或胰蛋白酶降解进行化学修饰。After the reaction mixture was diluted 2-5 times, it was added to RP-HPLC. HPLC condition: C4 column; Flow rate 10ml/min; A buffer solution: the aqueous solution containing 10% ACN and 0.1% TFA; B buffer solution: the ACN solution containing 0.1% TFA; Linear gradient: B% 0-40% (0- 80 minutes); PEG-insulin or PEG-insulin analog was collected at about 35% B. Desired compounds were confirmed by MAIDI-TOF, followed by chemical modification by sulfite hydrolysis or trypsin degradation.
实施例3Example 3
胰岛素受体结合测定：Insulin receptor binding assay:
在竞争结合测定中，采用闪烁亲近技术，测定了每种肽对胰岛素或IGF-1受体的亲和力。在Tris-Cl缓冲液(0.05M Tris-HCl(pH 7.5)、0.15M NaCl、0.1％w/v牛血清白蛋白)中对肽进行系列的3倍稀释，然后在96孔板(Corning Inc.，Acton，MA)中与0.05nM(3-[125I]-碘酪氨酰基)A TyrA14胰岛素或(3-[125I]-碘酪氨酰基)IGF-1(AmershamBiosciences，Piscataway，NJ)混合。将从过量表达人胰岛素或IGF-1受体的细胞中制备的等分量的1-6微克质膜片段加入各孔中，加入0.25mg/孔聚乙烯亚胺处理的麦胚凝集素A型闪烁亲近测定珠粒(Amersham Biosciences，Piscataway，NJ)。以800rpm振摇5分钟后，将板于室温保温12小时，用MicroBeta1450液体闪烁计数器(Perkin-Elmer，Wellesley，MA)测量放射性。在各孔中，用浓度比试验样品最高浓度高4倍的过量“非放射性”天然配体测量非特异性结合(NSB)的放射性。在无竞争剂的各孔中检测总的结合放射性。如下计算百分比特异性结合：％特异性结合＝(结合的NSB/总的结合的NSB)x100。应用Origin软件(OriginLab，Northampton，MA)求出IC50值。The affinity of each peptide for the insulin or IGF-1 receptor was determined using the scintillation proximity technique in a competition binding assay. Peptides were serially diluted 3-fold in Tris-Cl buffer (0.05M Tris-HCl (pH 7.5), 0.15M NaCl, 0.1% w/v bovine serum albumin) and then plated in 96-well plates (Corning Inc. , Acton, MA) was mixed with 0.05 nM (3-[125I]-iodotyrosyl)A TyrA14 insulin or (3-[125I]-iodotyrosyl)IGF-1 (Amersham Biosciences, Piscataway, NJ). Aliquots of 1-6 micrograms of plasma membrane fragments prepared from cells overexpressing human insulin or IGF-1 receptors were added to each well, and 0.25 mg/well polyethyleneimine-treated wheat germ agglutinin A-type scintillation was added Proximity assay beads (Amersham Biosciences, Piscataway, NJ). After shaking at 800 rpm for 5 minutes, the plate was incubated at room temperature for 12 hours and radioactivity was measured with a MicroBeta 1450 liquid scintillation counter (Perkin-Elmer, Wellesley, MA). In each well, non-specifically bound (NSB) radioactivity was measured with an excess of "non-radioactive" natural ligand at a concentration 4-fold higher than the highest concentration of the test sample. Total bound radioactivity was measured in wells without competitor. Percent specific binding was calculated as follows: % specific binding = (NSB bound/total NSB bound) x 100. IC50 values were calculated using Origin software (OriginLab, Northampton, MA).
实施例4Example 4
胰岛素受体磷酸化测定：Insulin receptor phosphorylation assay:
为了测量胰岛素或胰岛素类似物的受体磷酸化，将受体转染的HEK293细胞接种到96孔组织培养板(Costar #3596，Cambridge，MA)中，并在补充了100IU/ml青霉素、100μg/ml链霉素、10mM HEPES和0.25％牛生长血清的Dulbecco改良的Eagle培养基(DMEM)(HyCloneSH30541，Logan，UT)中在37℃、5％CO2和90％湿度下培养16-20小时。在补充了0.5％牛血清白蛋白的DMEM(Roche Applied Science #100350，Indianapolis，IN)中制备系列稀释的胰岛素或胰岛素类似物，并与贴壁细胞一起加入各孔中。在潮湿环境与5％CO2下于37℃温育15分钟后，细胞用5％低聚甲醛于室温固定20分钟，用磷酸缓冲盐溶液(pH 7.4)洗涤两次，用2％牛血清白蛋白/PBS封闭1小时。然后，将板洗涤三次，加入按照生产商的建议在PBS与2％牛血清白蛋白复溶的针对磷酸酪氨酸的辣根过氧化物酶缀合的抗体(Upstatebiotechnology #16-105，Temecula，CA)。在室温下保温温3小时后，将板洗涤4次，将0.1mlTMB单一溶液底物(Invitrogen，#00-2023，Carlbad，CA)加入各孔中。5分钟后通过加入0.05ml 1N HCl停止显色。在Titertek Multiscan MCC340(ThermoFisher，Pittsburgh，PA)中测量450nm下的吸光度。绘制吸光度相对于肽浓度的剂量反应曲线，应用Origin软件(OriginLab，Northampton，MA)求出EC50值。To measure receptor phosphorylation by insulin or insulin analogues, receptor-transfected HEK293 cells were seeded into 96-well tissue culture plates (Costar #3596, Cambridge, MA) in the presence of 100 IU/ml penicillin, 100 μg/ml ml streptomycin, 10 mM HEPES, and 0.25% bovine growth serum in Dulbecco's Modified Eagle Medium (DMEM) (HyClone SH30541, Logan, UT) for 16-20 hours at 37° C., 5% CO2 and 90% humidity. Serial dilutions of insulin or insulin analogs were prepared in DMEM supplemented with 0.5% bovine serum albumin (Roche Applied Science #100350, Indianapolis, IN) and added to each well along with adherent cells. After incubation at 37°C for 15 minutes in a humid environment with 5% CO2 , the cells were fixed with 5% paraformaldehyde at room temperature for 20 minutes, washed twice with phosphate buffered saline (pH 7.4), and whitened with 2% bovine serum. Protein/PBS blocking for 1 hour. Then, the plate was washed three times, and a horseradish peroxidase-conjugated antibody against phosphotyrosine (Upstatebiotechnology #16-105, Temecula, CA). After incubation at room temperature for 3 hours, the plate was washed 4 times and 0.1 ml of TMB single solution substrate (Invitrogen, #00-2023, Carlbad, CA) was added to each well. Color development was stopped after 5 minutes by adding 0.05 ml 1N HCl. Absorbance at 450 nm was measured in a Titertek Multiscan MCC340 (ThermoFisher, Pittsburgh, PA). A dose-response curve of absorbance versus peptide concentration was plotted, and the EC50 value was calculated using Origin software (OriginLab, Northampton, MA).
实施例5Example 5
确定模型二肽裂解(PBS中)的速率Determining the rate of model dipeptide cleavage (in PBS)
将一种特殊的六肽(HSRGTF-NH2；SEQ ID NO：59)用作模型肽，在该模型肽上可研究二肽N端延伸物的裂解速率。制备二肽延伸的模型肽，将Boc保护的肌氨酸和赖氨酸相继加入模型肽结合的树脂上以产生肽A(Lys-Sar-HSRGTF-NH2；SEQ ID NO：60)。通过HF使肽A裂解后，用制备型HPLC纯化。A specific hexapeptide (HSRGTF-NH2 ; SEQ ID NO: 59) was used as a model peptide on which the cleavage rate of the dipeptide N-terminal extension could be studied. To prepare a dipeptide-extended model peptide, Boc-protected sarcosine and lysine were sequentially added to the model peptide-bound resin to generate peptide A (Lys-Sar-HSRGTF-NH2 ; SEQ ID NO: 60). After peptide A was cleaved by HF, it was purified by preparative HPLC.
测定相应前肽的裂解速率。通过其相应的峰面积求出前肽和模型母体肽的浓度。通过绘制不同时间间隔下前药浓度的对数曲线图，来求出前药的一级解离速率常数。该曲线的斜率给出速率常数‘k’。用式t1/2＝.693/k，计算各种前药裂解的半寿期。测出该模型肽HSRGTF-NH2(SEQ ID NO：59)的Lys-Sar延伸物的半寿期为14.0小时。The cleavage rate of the corresponding propeptide was determined. Concentrations of propeptide and model parent peptide were determined from their corresponding peak areas. The first order dissociation rate constant of the prodrug is obtained by plotting the logarithmic curve of the prodrug concentration at different time intervals. The slope of this curve gives the rate constant 'k'. Using the formula t1/2 =.693/k, the cleavage half-lives of various prodrugs were calculated. The half-life of the Lys-Sar extension of the model peptide HSRGTF-NH2 (SEQ ID NO: 59) was determined to be 14.0 hours.
实施例6Example 6
用全d-同工型模型肽测定的血浆中二肽裂解半寿期的速率Rate of Dipeptide Cleavage Half-Life in Plasma Determined with the All D-Isoform Model Peptide
使用另外的模型六肽(dHdTdRGdTdF-NH2SEQIDNO：63)测定血浆中二肽裂解的速率。使用各氨基酸的d-异构体以防止除前药延伸物以外的模型肽的酶促切割。这种模型d-异构体六肽以类似于l-异构体的方式合成。如先前对肽A所报道，将肌氨酸和赖氨酸相继加至N端以制备肽B(dLys-dSar-dHdTdRGdTdF-NH2SEQ ID NO：64)。The rate of dipeptide cleavage in plasma was determined using an additional model hexapeptide (dHdTdRGdTdF-NH2 SEQ ID NO: 63). The d-isomer of each amino acid was used to prevent enzymatic cleavage of model peptides other than prodrug extenders. This model d-isomer hexapeptide was synthesized in a similar manner to the l-isomer. As previously reported for peptide A, sarcosine and lysine were sequentially added to the N-terminus to prepare peptide B (dLys-dSar-dHdTdRGdTdF-NH2 SEQ ID NO: 64).
测定各个前肽的裂解速率。通过其各自的峰面积求出前肽和模型母体肽的浓度。通过绘制不同时间间隔下前药浓度的对数曲线图，来求出前药的一级解离速率常数。该曲线的斜率给出速率常数‘k’。测出这个模型肽dHdTdRGdTdF-NH2(SEQ ID NO：63)的Lys-Sar延伸物的半寿期为18.6小时。The rate of cleavage of each propeptide was determined. Concentrations of propeptide and model parent peptide were determined from their respective peak areas. The first order dissociation rate constant of the prodrug is obtained by plotting the logarithmic curve of the prodrug concentration at different time intervals. The slope of this curve gives the rate constant 'k'. The half-life of the Lys-Sar extension of this model peptide dHdTdRGdTdF-NH2 (SEQ ID NO: 63) was determined to be 18.6 hours.
实施例7Example 7
采用实施例5中所述方法，测定与模型六肽(HSRGTF-NH2；SEQ ID NO：59)连接的其它二肽的裂解速率。在这些实验中得到的结果见表2和表3。Using the method described in Example 5, the rate of cleavage of other dipeptides linked to the model hexapeptide (HSRGTF-NH2 ; SEQ ID NO: 59) was determined. The results obtained in these experiments are shown in Tables 2 and 3.
表2：PBS中与N端对氨基-Phe侧链连接的二肽O-U自模型六肽(HSRGTF-NH2；SEQ IDNO：59)的的裂解Table 2: Cleavage of dipeptide OU from a model hexapeptide (HSRGTF-NH2 ; SEQ ID NO: 59) linked to the N-terminal para-amino-Phe side chain in PBS
表3：PBS中与1位(X)上的组氨酸(或组氨酸衍生物)连接的二肽U-O自模型六肽(XSRGTF-NH2；SEQ ID NO：59)的裂解Table 3: Cleavage of the dipeptide UO linked to histidine (or histidine derivatives) at position 1 (X) from a model hexapeptide (XSRGTF-NH2 ; SEQ ID NO:59) in PBS
NH2-U-O-XSRGTF-NH2NH2 -UO-XSRGTF-NH2
实施例8Example 8
鉴定结构适于前药构建的胰岛素类似物Identification of insulin analogs structurally amenable to prodrug construction
已知A链的19位是胰岛素活性的重要部位。因此，在该部位进行修饰以允许前药元件的连接是理想的。已合成了胰岛素在A19上的特定类似物，并表征了其对胰岛素受体的活性。在A19上鉴定出两种高活性结构类似物，其中在第二活性部位芳族残基(B24)上的相当结构变化在鉴定类似完整活性的胰岛素类似物方面是不成功的。It is known that position 19 of the A chain is an important site for insulin activity. Therefore, modifications at this site to allow attachment of prodrug elements are desirable. Specific analogs of insulin at A19 have been synthesized and characterized for their activity at the insulin receptor. Two highly active structural analogs were identified on A19, where comparable structural changes at the second active site aromatic residue (B24) were unsuccessful in identifying insulin analogs with similar full activity.
表4和表5阐述对于针对胰岛素受体的完整活性，A19位具有高度结构保守性(用实施例3所述测定法测定受体结合)。表4表明只有两种在A19上具有修饰的胰岛素类似物具有类似于天然胰岛素的受体结合活性。对于4-氨基胰岛素类似物，提供了三个单独实验的数据。标示“活性(试验中)”的一栏比较了同时进行的两个单独实验的胰岛素类似物相对于天然胰岛素的结合百分比。标示“活性(0.60nM)”的一栏是胰岛素类似物相对于使用该测定法得到的胰岛素结合的历史平均值的相对结合百分比。在任一种分析中，两种A19胰岛素类似物(4-氨基苯丙氨酸和4-甲氧基苯丙氨酸)显示大致等于天然胰岛素的受体结合。图3表示显示天然胰岛素和A19胰岛素类似物对胰岛素受体的各自特异性结合的曲线图。表5提供的数据表明，具有与天然胰岛素相当的结合活性的两种A19胰岛素类似物(4-氨基和4-甲氧基)，还显示对胰岛素受体相当的活性(采用实施例4所述测定法测定受体活性)。Tables 4 and 5 illustrate that the A19 position is highly structurally conserved for full activity against the insulin receptor (receptor binding determined using the assay described in Example 3). Table 4 shows that only two insulin analogues with modifications at A19 have receptor binding activity similar to native insulin. For the 4-amino insulin analogs, data from three separate experiments are presented. The column labeled "Activity (in assay)" compares the percent binding of the insulin analog relative to native insulin from two separate experiments performed simultaneously. The column labeled "Activity (0.60 nM)" is the relative percent binding of the insulin analog relative to the historical average of insulin binding using this assay. In either assay, two A19 insulin analogs (4-aminophenylalanine and 4-methoxyphenylalanine) showed receptor binding approximately equal to native insulin. Figure 3 represents a graph showing the respective specific binding of native insulin and A19 insulin analogues to the insulin receptor. The data provided in Table 5 show that two A19 insulin analogs (4-amino and 4-methoxyl), which have comparable binding activity to native insulin, also exhibit comparable activity on the insulin receptor (using the method described in Example 4). assay to measure receptor activity).
表4：A19胰岛素类似物的胰岛素受体结合活性Table 4: Insulin receptor binding activity of A19 insulin analogs
表5：A19胰岛素类似物的胰岛素受体磷酸化活性Table 5: Insulin receptor phosphorylation activity of A19 insulin analogs
实施例9Example 9
胰岛素样生长因子(IGF)类似物Insulin-like Growth Factor (IGF) Analogs
用于分离胰岛素A链的典型纯化方案使用NH4HCO3缓冲液(pH＝7.8)。在这些条件下，二肽前药元件快速从A链上裂解。为了简化前药的纯化以研究其对胰岛素受体的活性，申请人使用IGF类似物进行了这类研究，所述IGF类似物显示与天然胰岛素的活性类似对胰岛素受体的活性。更具体地讲，IGF类似物(IGF1(YB16LB17)包含天然IGF A链和B链(分别为SEQ ID NO：61和SEQ ID NO：62)，其中天然IGF B链的15和16位(分别相当于天然胰岛素B链的16和17位)上的天然谷氨酰胺和苯丙氨酸已分别用酪氨酸和亮氨酸残基置换。如图4和下表6所示，IGF1(YB16LB17)的结合活性表明，所述化合物是十分有效的胰岛素类似物。A typical purification protocol for isolation of the insulinA chain usesNH4HCO3 buffer (pH=7.8). Under these conditions, the dipeptide prodrug element is rapidly cleaved from the A chain. In order to simplify the purification of prodrugs to study their activity on the insulin receptor, applicants performed such studies using IGF analogues which showed an activity on the insulin receptor similar to that of native insulin. More specifically, the IGF analog (IGF1 (YB16 LB17 ) comprises native IGF A chain and B chain (SEQ ID NO: 61 and SEQ ID NO: 62, respectively), wherein the 15 and 16 positions of the native IGF B chain (respectively corresponding to the 16 and 17 positions of the natural insulin B chain) the natural glutamine and phenylalanine have been replaced with tyrosine and leucine residues, respectively.As shown in Figure 4 and Table 6 below, IGF1 The binding activity of (YB16 LB17 ) indicates that the compound is a very potent insulin analog.
表6Table 6
实施例10Example 10
IGF前药衍生物IGF Prodrug Derivatives
根据A19胰岛素类似物的活性(参见实施例5)，对IGF1A:B(YB16LB17)类似物进行了类似修饰，并研究了其结合和刺激胰岛素受体活性的能力。图5提供以下通用合成流程，其用于制备IGF1A:B(YB16LB17)(其中天然酪氨酸用4-氨基苯丙氨酸置换[IGF1A:B(YB16LB17)(p-NH2-F)A19酰胺])，以及用于制备其二肽延伸的衍生物[IGF1 A:B(YB16LB17)A19-AiBAla酰胺](其中包含AiB和Ala的二肽通过与A19 4-氨基苯丙氨酸的酰胺键与所述肽连接)。如图6和表7所示，IGF类似物IGF1(YB16LB17)A(p-NH2-F)19与胰岛素受体特异性结合，其中该类似物的二肽延伸衍生物不能与胰岛素受体特异性结合。注意到二肽延伸物缺乏可供自发裂解二肽的适当结构(在二肽的第二个位置上不存在N-烷基化氨基酸)，因此不存在胰岛素受体结合的恢复。Based on the activity of the A19 insulin analog (see Example 5), the IGF1A:B(YB16 LB17 ) analog was similarly modified and studied for its ability to bind and stimulate insulin receptor activity. Figure 5 provides the following general synthetic scheme for the preparation of IGF1A:B(YB16 LB17 ) (wherein the natural tyrosine is replaced with 4-aminophenylalanine [IGF1A:B(YB16 LB17 )(p-NH2 -F)A19 amide]), and for the preparation of its dipeptide-extended derivative [IGF1 A:B(YB16 LB17 )A19 -AiBAla amide] (wherein the dipeptide containing AiB and Ala is obtained by combining with A19 4- The amide bond of the aminophenylalanine is attached to the peptide). As shown in Figure 6 and Table 7, the IGF analogue IGF1(YB16 LB17 )A(p-NH2 -F)19 specifically binds to the insulin receptor, and the dipeptide extended derivative of this analogue cannot bind to insulin Receptor specific binding. Note that the dipeptide extension lacks the proper structure for spontaneous cleavage of the dipeptide (absence of N-alkylated amino acid at the second position of the dipeptide) and thus there is no restoration of insulin receptor binding.
表7Table 7
制备IGFB16B17衍生肽的又一种前药衍生物，其中二肽前药元件(丙氨酸-脯氨酸)通过酰胺键与A链氨基端连接(IGF1(YB16LB17)(AlaPro)A-1，0)。如表8所示，IGF1(YB16LB17)(AlaPro)A-1，0对胰岛素受体的亲和力显著下降。根据表3数据，注意到二肽前药元件缺乏可供自发裂解二肽前药元件的适当结构，因此检测到的胰岛素受体结合不是前药元件裂解的结果。Preparation of yet another prodrug derivative of the IGFB16B17- derived peptide, wherein the dipeptide prodrug element (alanine-proline) is linked to the amino terminus of the A chain through an amide bond (IGF1(YB16 LB17 )(AlaPro)A -1,0 ). As shown in Table 8, the affinity of IGF1(YB16 LB17 )(AlaPro)A-1,0 to insulin receptor was significantly decreased. From the data in Table 3, it is noted that the dipeptide prodrug element lacks an appropriate structure for spontaneous cleavage of the dipeptide prodrug element, and thus the detected insulin receptor binding is not the result of cleavage of the prodrug element.
表8Table 8
实施例11Example 11
其它的IGF胰岛素类似物。Other IGF insulin analogs.
IGF1(YB16LB17)(YL)B16B17肽序列的进一步修饰披露了对胰岛素和IGF-1受体的效能不同的其它IGF胰岛素类似物。这些类似物中每种的结合数据见表9(采用实施例3的测定法)，其中修饰的位置根据天然胰岛素肽(DPI＝des B26-30)的相应位置指定。例如，本文提及的缺乏任何更多详尽描述的“位置B28”可能意指其中SEQ ID NO：2的第一氨基酸已缺失的胰岛素类似物B链的相应B27位。因此，一般提及的“B(Y16)”是指天然IGF-1序列B链15位上的酪氨酸残基的取代。有关胰岛素和IGF类似物的相对受体结合的数据见表9，有关IGF类似物刺激的磷酸化(采用实施例4的测定法)的数据见表10。Further modification ofthe IGF1(YB16LB17 )(YL)B16B17 peptide sequence revealed other IGF insulin analogs with different potenciesat the insulin and IGF-1 receptors. Binding data for each of these analogs is presented in Table 9 (using the assay of Example 3), where the positions of the modifications are assigned relative to the corresponding positions of the native insulin peptide (DPI = des B26-30). For example, reference herein to "position B28" lacking any further elaboration may mean the corresponding position B27 of the B chain of an insulin analogue in which the first amino acid of SEQ ID NO: 2 has been deleted. Therefore, the general reference to "B(Y16)" refers to the substitution of the tyrosine residue at position 15 of the B chain of the native IGF-1 sequence. See Table 9 for data on relative receptor binding of insulin and IGF analogs, and Table 10 for IGF analog-stimulated phosphorylation (using the assay of Example 4).
实施例12Example 12
形成自IGFB16B17衍生肽的前药形式的二肽裂解Dipeptide cleavage to form prodrug forms from IGFB16B17-derived peptides
测定了(pNH2-Phe)酰胺连接的二肽AibPro从不同IGF-1肽上的裂解以确定肽序列或异源双链体对二肽裂解的影响。受测试肽的结果见表12，数据表明，仅IGF1-A链就代表研究IGF1 B:A(YL)B16，17肽的前药半寿期的良好模型。Cleavage of (pNH2-Phe) amide-linked dipeptide AibPro from different IGF-1 peptides was assayed to determine the effect of peptide sequence or heteroduplex on dipeptide cleavage. The results for the tested peptides are presented in Table 12, and the data indicate that the IGF1-A chain alone represents a good model for studying the prodrug half-life of the IGF1 B:A(YL)B16,17 peptide.
表12Table 12
对于前药形式，IGF A链的前药衍生物与二硫化物结合的A链和B链构建体(IGF1A:B(YB16LB17)的比较显示，两种化合物具有类似的半寿期。因此，经测定仅IGF1A链就是研究IGF1 B:A(YB16LB17)上的前药半寿期的良好模型。注意，AibAla衍生物不裂解，因此不是前药，但用来表明修饰可使胰岛素类似物IGF1 A:B(YB16LB17)(p-NH2-F)A19酰胺钝化。为简单起见，使用不存在B链的仅IGF1 A链来测定前药半寿期。各前肽的半寿期按实施例5所述方法测定。数据见表13：For the prodrug form, comparison of prodrug derivatives of the IGF A chain with disulfide-conjugated A and B chain constructs (IGF1A:B(YB16 LB17 ) revealed similar half-lives for both compounds. Therefore, the IGF1A chain alone was determined to be a good model to study the half-life of prodrugs on IGF1 B:A(YB16 LB17 ). Note that the AibAla derivative is not cleaved and therefore not a prodrug, but is used to show that modifications can make Insulin analogue IGF1 A:B(YB16 LB17 )(p-NH2 -F)A19 amide inactivation. For simplicity, the prodrug half-life was determined using only the IGF1 A chain in the absence of the B chain. The half-life of the peptide was measured by the method described in Example 5. The data are shown in Table 13:
表13：IGF1二肽延伸的(p-NH2-F)A19酰胺上的二肽半寿期Table 13: Dipeptide half-life on IGF1 dipeptide extended (p-NH2 -F)A19 amide
数据表明，通过改变二肽前药元件上的取代基，前药的半寿期可从2小时到＞100小时不等。The data indicate that by varying the substituents on the dipeptide prodrug element, the half-life of the prodrug can vary from 2 hours to >100 hours.
使用IGF1-A(pNH2-F)19型肽并改变通过A19位上4-氨基苯丙氨酸连接的二肽前药元件的氨基酸组成，来制备其它前药衍生肽。在PBS和在20％血浆/PBS(即存在血清酶时)中测定不同构建体的二肽半寿期。结果见表14。结果表明经测定的4种肽中有3种不受血清酶的影响。Other prodrug-derived peptides were prepared using the IGF1-A(pNH2-F) type19 peptide and varying the amino acid composition of the dipeptide prodrug element linked through the 4-aminophenylalanine at position A19. The dipeptide half-life of the different constructs was determined in PBS and in 20% plasma/PBS (ie in the presence of serum enzymes). The results are shown in Table 14. The results showed that 3 of the 4 peptides tested were not affected by serum enzymes.
表14：IGF1-A(pNH2-F)19上的二肽半寿期Table 14: Dipeptide half-lives on IGF1-A(pNH2-F)19
实施例13Example 13
IGFB16B17衍生肽随时间变化的受体结合Time-dependent receptor binding of IGFB16B17- derived peptides
制备IGFB16B17衍生肽的前药制剂，并使用实施例3的胰岛素受体结合测定法测定其随时间变化的降解。如下制备用于该测定法的肽：Prodrug formulations of IGFB16B17- derived peptides were prepared and their time-dependent degradation was determined using the insulin receptor binding assay of Example 3. Peptides for this assay were prepared as follows:
二肽-IGF1A类似物Dipeptide-IGF1A analogs
如无具体说明，则Boc-化学法用于指定肽类似物的合成。将选定的二肽H2N-AA1-AA2-COOH加到IGF1A(Ala)6，7，11，20上的(pNH2-Phe)19。在MBHA树脂上合成IGF-1 A链C端三肽Boc(Fmoc-pNH-Phe)-Ala-Ala。用20％哌啶/DMF于室温处理30分钟脱去Fmoc后，使用3倍过量的氨基酸、PyBop、DIEA和催化量的吡啶，使Fmoc-AA2与在A19上的对氨基苄基侧链偶联。采用Applied Biosystems 430A肽合成仪，完成其余IGF-1A链(Ala)6，7，11，20序列的Boc-合成，得到IGF-1 A链(Boc)0(Ala)6，7，11，20(Fmoc-AA2-pNH-Phe)19-MBHA。在从AA2的N端脱去Fmoc基团后，使用3倍过量的氨基酸、DEPBT和DIEA使Boc-AA1与胺偶联。通过TFA脱去余留在A链上的两个Boc基团，接着通过HF裂解，得到IGF-1A链(Ala)6，7，11，20(H2N-AA1-AA2-pNH-Phe)19酰胺。在AA1为d-赖氨酸的情况下，在ε-胺上进行乙酰化后脱去Boc。二肽-IGF-1 A链类似物用半制备型RP-HPLC纯化，并用通过分析型RP-HPLC和MALDI质谱法表征。If not specified, Boc-chemistry was used for the synthesis of the indicated peptide analogs. Addition of selected dipeptides H2N-AA1-AA2 -COOH to (pNH2- Phe)19 on IGF1A(Ala)6,7,11,20 . The C-terminal tripeptide Boc(Fmoc-pNH-Phe)-Ala-Ala of IGF-1 A chain was synthesized on MBHA resin. After removal of Fmoc with 20% piperidine/DMF at room temperature for 30 min, Fmoc-AA2 was coupled to the p-aminobenzyl side chain on A19 using a 3-fold excess of amino acid, PyBop, DIEA, and a catalytic amount of pyridine . Using Applied Biosystems 430A peptide synthesizer, complete the Boc-synthesis of the remaining IGF-1A chain (Ala) 6, 7, 11,20 sequences to obtain IGF-1 A chain (Boc)0 (Ala) 6, 7, 11,20 (Fmoc-AA2-pNH-Phe)19 -MBHA. After removal of the Fmoc group from the N-terminus of AA2, Boc-AA1 was coupled to the amine using a 3-fold excess of amino acid, DEPBT and DIEA. The remaining two Boc groups on chain A were removed by TFA, followed by cleavage by HF to obtain IGF-1A chain (Ala) 6, 7, 11, 20 (H2 N-AA1-AA2-pNH -Phe)19 amides. In the case of AA1 being d-lysine, Boc is removed after acetylation on the ε-amine. The dipeptide-IGF-1 A chain analog was purified by semi-preparative RP-HPLC and characterized by analytical RP-HPLC and MALDI mass spectrometry.
二肽-IGF-1(YL)类似物Dipeptide-IGF-1(YL) analogs
如紧接的上文中所述但将PAM树脂用于合成IGF-1A链以在HF裂解时得到C端酸，将选定的二肽H2N-AA1-AA2-COOH加到IGF-1 A链(Acm)6，11，20上的(pNH2-Phe)19。在MBHA树脂上合成IGF-1 B链(YL)16，17(Acm)19，得到C端酰胺。在100％DMSO中通过与DTNP按1∶1摩尔比率反应，用Npys修饰CysB7上的游离巯基。采用流程1所示的“1+2”两步链组合策略，装配经纯化的二肽-IGF-1 A链和IGF-1 B链(YL)16，17衍生物。在半制备型RP-HPLC进行中间纯化和最终纯化，并用分析型RP-HPLC和MALDI质谱法表征。Add the selected dipeptide H2N-AA1-AA2 -COOH to IGF-1 A as described immediately above but using PAM resin for IGF-1A chain synthesis to give C-terminal acid upon HF cleavage (pNH2 -Phe)19 on chain (Acm)6,11,20 . IGF-1 B chain (YL)16,17 (Acm)19 was synthesized on MBHA resin to obtain the C-terminal amide. The free sulfhydryl group on CysB7 was modified with Npys by reaction with DTNP in 1:1 molar ratio in 100% DMSO. Purified dipeptide-IGF-1 A chain and IGF-1 B chain (YL ) derivatives were assembled using the "1+2" two-step chain combination strategy shown in Scheme 1. Intermediate and final purifications were performed on semi-preparative RP-HPLC and characterized by analytical RP-HPLC and MALDI mass spectrometry.
将IGFB16B17衍生肽前药在PBS(pH 7.4)中于37℃保温，在预定的时间间隔内取出等分量，进一步的降解用0.1％TFA淬灭，对等分量进行分析型HPLC分析。用LC-MS鉴定代表IGFB16B17衍生肽的前药形式和活性形式的a峰和b峰，并通过HPLC峰面积的积分进行量化。图8A-8C显示IGFB16B17衍生肽前药：IGF1A(Ala)6，7，11，20(Aib-Pro-pNH-F)19降解的HPLC分析输出结果。在开始将前药于PBS中保温后20分钟(图8A)、81分钟(图8B)和120分钟(图8C)内取出等分量。数据表明IGF1A(Ala)6，7，11，20(Aib-Pro-pNH-F)19酰胺随时间自发性非酶促转化为IGF1A(Ala)6，7，11，20(pNH2-F)1酰胺。The IGFB16B17- derived peptide prodrug was incubated in PBS (pH 7.4) at 37°C, aliquots were taken at predetermined time intervals, further degradation was quenched with 0.1% TFA, and aliquots were analyzed by analytical HPLC. Peaks a and b representing the prodrug and active forms of the IGFB16B17 -derived peptide were identified by LC-MS and quantified by integration of the HPLC peak areas. Figures 8A-8C show the output of HPLC analysis of the degradation of IGFB16B17 derived peptide prodrug: IGF1A(Ala)6,7,11,20 (Aib-Pro-pNH-F)19 . Aliquots were withdrawn at 20 minutes (Figure 8A), 81 minutes (Figure 8B) and 120 minutes (Figure 8C) after starting the incubation of the prodrug in PBS. Data indicate spontaneous non-enzymatic conversion of IGF1A(Ala)6,7,11,20 (Aib-Pro-pNH-F)19 amide to IGF1A(Ala)6,7,11,20 (pNH2 -F) over time1 amide.
用实施例3的体外测定法测量，根据化合物结合胰岛素受体的能力，还测定了IGFB16B17衍生肽的前药形式向其活性形式的降解。图9A和图9B曲线图表示前药Aib，dPro-IGF1YL(通过A194-氨基Phe连接的二肽)的体外活性。图9A曲线图比较了在PBS中温育的天然胰岛素(于4℃在1小时时测定)和A19IGF前药类似物(Aib，dPro-IGF1YL)随时间变化(0小时、2.5小时和10.6小时)的相对胰岛素受体结合。图9B曲线图比较了在20％血浆/PBS中保温的天然胰岛素(于4℃在1.5小时时测定)和A19IGF前药类似物(Aib，dPro-IGF1YL)随时间变化(0小时、1.5小时和24.8小时)的相对胰岛素受体结合。图中所提供的数据表明，随着前药形式转化为活性IGF1YL肽，从A19IGF前药类似物样品恢复的活性渐增。针对胰岛素受体结合测定了IGFB16B17衍生肽的活性，由于潜在的IGFB16B17衍生肽的活性比天然胰岛素的活性高，因此相对于胰岛素大于100％的活性是可能的。Degradation of the prodrug form of the IGFB16B17- derived peptide to its active form was also determined based on the ability of the compound to bind the insulin receptor as measured by the in vitro assay of Example 3. Figures 9A and 9B are graphs showing the in vitro activity of the prodrug Aib, dPro-IGF1YL (dipeptide linked through A194-aminoPhe). Fig. 9 A graph compares the natural insulin (measured at 4 ℃ at 1 hour) and A19IGF prodrug analog (Aib, dPro-IGF1YL) in PBS incubated in PBS over time (0 hour, 2.5 hours and 10.6 hours). Relative insulin receptor binding. Figure 9B is a graph comparing the natural insulin (measured at 4°C at 1.5 hours) and A19IGF prodrug analogs (Aib, dPro-IGF1YL) over time (0 hours, 1.5 hours and 24.8 hours) relative insulin receptor binding. The data presented in the figure show that the recovery of activity from the A19IGF prodrug analog samples is progressive as the prodrug form is converted to the active IGF1YL peptide. The activity of the IGFB16B17- derived peptides was determined for insulin receptor binding, and since the activity of the potential IGFB16B17- derived peptides is higher than that of native insulin, an activity of greater than 100% relative to insulin is possible.
图10A和图10B曲线图表示前药dK，(N-异丁基G)-IGF1YL(通过A194-氨基Phe连接的二肽)的体外活性。图10A曲线图比较了在PBS中保温的天然胰岛素(于4℃在1小时时测定)和A19IGF前药类似物(IGF1YL:dK，(N-异丁基G)随时间变化(0小时、5小时和52小时)的相对胰岛素受体结合。图10B曲线图比较了在20％血浆/PBS中保温的天然胰岛素(于4℃在1.5小时时测定)和A19IGF前药类似物(IGF1YL:dK，(N-异丁基G)随时间变化(0小时、3.6小时和24.8小时)的相对胰岛素受体结合。图中所提供的数据表明，随着前药形式转化为活性IGF1YL肽，从A19IGF前药类似物样品恢复的活性渐增。Figures 10A and 10B are graphs showing the in vitro activity of the prodrug dK,(N-isobutylG)-IGF1YL (dipeptide linked through A194-aminoPhe). Figure 10A graph compares the natural insulin (measured at 4 ℃ at 1 hour) and the A19IGF prodrug analog (IGF1YL:dK, (N-isobutyl G) over time (0 hour, 5 hours) incubated in PBS. hours and 52 hours) relative insulin receptor binding. Figure 10B graph compares native insulin (measured at 4°C at 1.5 hours) and A19IGF prodrug analogs (IGF1YL:dK, (N-isobutyl G) relative insulin receptor binding as a function of time (0 hours, 3.6 hours and 24.8 hours). The data presented in the figure show that the conversion from the A19IGF pro The drug analog samples recovered increasing activity.
图11A和图11B曲线图表示前药dK(e-乙酰基)，Sar)-IGF1YL(通过A19 4-氨基Phe连接的二肽)的体外活性。图11A曲线图比较了在PBS中保温的天然胰岛素(于4℃在1小时时测定)和A19 IGF前药类似物(IGF1YL:dK(e-乙酰基)，Sar)随时间变化(0小时、7.2小时和91.6小时)的相对胰岛素受体结合。图11B曲线图比较了在20％血浆/PBS中保温的天然胰岛素(于4℃在1.5小时时测定)和A19IGF前药类似物(IGF1YL:dK(e-乙酰基)，Sar)随时间变化(0小时、9小时和95小时)的相对胰岛素受体结合。图中所提供的数据表明，随着前药形式转化为活性IGF1YL肽时，从A19IGF前药类似物样品恢复的活性渐增。Figures 11A and 11B are graphs showing the in vitro activity of the prodrug dK(e-acetyl),Sar)-IGF1YL (dipeptide linked through A19 4-aminoPhe). Figure 11A graph compares the natural insulin (measured at 4 ℃ at 1 hour) and the A19 IGF prodrug analogue (IGF1YL:dK(e-acetyl), Sar) over time (0 hour, 0 hour, Sar) incubated in PBS. 7.2 hours and 91.6 hours) of relative insulin receptor binding. Figure 11B graph compares the natural insulin (measured at 4°C at 1.5 hours) and the A19IGF prodrug analog (IGF1YL: dK(e-acetyl), Sar) over time in 20% plasma/PBS incubation ( 0 hr, 9 hr and 95 hr) relative insulin receptor binding. The data presented in the figure demonstrates that the recovery of activity from the A19IGF prodrug analog samples is progressive as the prodrug form is converted to the active IGF1YL peptide.
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| CN102245624A CN102245624A (en) | 2011-11-16 |
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| EP (1) | EP2376521B1 (en) |
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|---|---|---|---|---|
| WO2009099763A1 (en) | 2008-01-30 | 2009-08-13 | Indiana University Research And Technology Corporation | Ester-based peptide prodrugs |
| CL2009001424A1 (en) | 2008-06-17 | 2010-04-30 | Univ Indiana Res & Tech Corp | Glucagon-like peptide; dimer comprising two of said peptides; pharmaceutical composition comprising it; and its use to treat diabetes or induce weight loss. |
| EA020537B1 (en) | 2008-12-15 | 2014-11-28 | Зилэнд Фарма А/С | Glucagon analogues |
| US8642540B2 (en) | 2008-12-15 | 2014-02-04 | Zealand Pharma A/S | Glucagon analogues |
| ES2502218T3 (en) | 2008-12-15 | 2014-10-03 | Zealand Pharma A/S | Glucagon analogues |
| CN102282166B (en) | 2008-12-15 | 2015-04-01 | 西兰制药公司 | Glucagon analogues |
| CN102325539A (en) | 2008-12-19 | 2012-01-18 | 印第安纳大学研究及科技有限公司 | Amide-based glucagon superfamily peptide prodrugs |
| PE20120914A1 (en) | 2009-06-16 | 2012-08-22 | Univ Indiana Res & Tech Corp | GIP RECEIVER ACTIVE GLUCAGON COMPOUNDS |
| EA022816B1 (en) | 2009-07-13 | 2016-03-31 | Зилэнд Фарма А/С | Acylated glucagon analogues |
| AR081975A1 (en) | 2010-06-23 | 2012-10-31 | Zealand Pharma As | GLUCAGON ANALOGS |
| WO2011160633A1 (en) | 2010-06-24 | 2011-12-29 | Zealand Pharma A/S | Glucagon analogues |
| WO2011163462A2 (en)* | 2010-06-24 | 2011-12-29 | Indiana University Research And Technology Corporation | Amide-based insulin prodrugs |
| EP2588127A4 (en)* | 2010-06-24 | 2014-06-11 | Univ Indiana Res & Tech Corp | MEDICINAL AGENTS RELATED TO A DIPEPTIDE |
| PH12013501495A1 (en) | 2011-01-20 | 2013-09-16 | Zealand Pharma As | Combination of acylated glucagon analogues with insulin analogues |
| US9187547B2 (en) | 2011-03-15 | 2015-11-17 | Novo Nordisk A/S | Human insulin analogues and derivatives comprising cysteine substitutions |
| EP2720711A1 (en)* | 2011-06-15 | 2014-04-23 | Novo Nordisk A/S | Multi substituted insulins |
| HRP20170890T1 (en) | 2011-06-22 | 2017-09-08 | Indiana University Research And Technology Corporation | Glucagon/glp-1 receptor co-agonists |
| CN104114183A (en)* | 2011-12-20 | 2014-10-22 | 印第安纳大学研究及科技有限公司 | CTP-based insulin analogs for treatment of diabetes |
| EA028665B1 (en) | 2012-05-03 | 2017-12-29 | Зилэнд Фарма А/С | Gip-glp-1 dual agonist compounds and methods |
| EP2875043B1 (en) | 2012-07-23 | 2016-12-21 | Zealand Pharma A/S | Glucagon analogues |
| TWI608013B (en) | 2012-09-17 | 2017-12-11 | 西蘭製藥公司 | Glucagon analog |
| EP2900255B1 (en)* | 2012-09-26 | 2019-01-30 | Indiana University Research and Technology Corporation | Insulin analog dimers |
| JP6735561B2 (en) | 2012-12-03 | 2020-08-05 | メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． | O-glycosylated carboxy-terminal portion (CTP) peptide-based insulin and insulin analogs |
| WO2014099577A1 (en) | 2012-12-17 | 2014-06-26 | Merck Sharp & Dohme Corp. | Process for purifying insulin and analogues thereof |
| CN105324397B (en) | 2013-03-14 | 2020-01-14 | 印第安纳大学研究及科技有限公司 | Insulin-incretin conjugates |
| US9427475B2 (en) | 2013-10-04 | 2016-08-30 | Merck Sharp & Dohme Corp. | Glucose-responsive insulin conjugates |
| US9988429B2 (en) | 2013-10-17 | 2018-06-05 | Zealand Pharma A/S | Glucagon analogues |
| AU2014336098B2 (en) | 2013-10-17 | 2018-05-10 | Boehringer Ingelheim International Gmbh | Acylated glucagon analogues |
| WO2015067715A2 (en) | 2013-11-06 | 2015-05-14 | Zealand Pharma A/S | Gip-glp-1 dual agonist compounds and methods |
| WO2015067716A1 (en) | 2013-11-06 | 2015-05-14 | Zealand Pharma A/S | Glucagon-glp-1-gip triple agonist compounds |
| US9822158B2 (en) | 2013-12-04 | 2017-11-21 | Merck Sharp & Dohme Corp. | Method for preparing crystalline insulin |
| EP3077008B1 (en) | 2013-12-06 | 2023-10-04 | Jie Han | Bioreversable promoieties for nitrogen-containing and hydroxyl-containing drugs |
| PE20161153A1 (en)* | 2014-01-20 | 2016-10-27 | Hanmi Pharm Ind Co Ltd | LONG-ACTING INSULIN AND USE OF THE SAME |
| AR099569A1 (en) | 2014-02-28 | 2016-08-03 | Novo Nordisk As | INSULIN DERIVATIVES AND THE MEDICAL USES OF THESE |
| US10407483B2 (en) | 2014-03-14 | 2019-09-10 | Merck Sharp & Dohme Corp. | Purifying insulin using cation exchange and reverse phase chromatography in the presence of an organic modifier and elevated temperature |
| AR100639A1 (en) | 2014-05-29 | 2016-10-19 | Hanmi Pharm Ind Co Ltd | COMPOSITION TO TREAT DIABETES THAT INCLUDES CONJUGATES OF PROLONGED INSULIN ANALOGS AND CONJUGATES OF PROLONGED INSULINOTROPIC PEPTIDES |
| TWI684458B (en) | 2014-05-30 | 2020-02-11 | 南韓商韓美藥品股份有限公司 | Composition for treating diabetes mellitus comprising insulin and a glp-1/glucagon dual agonist |
| WO2016014325A1 (en) | 2014-07-21 | 2016-01-28 | Merck Sharp & Dohme Corp. | Chromatography process for purification of inlsulin and insulin analogs |
| JP6657230B2 (en) | 2014-09-24 | 2020-03-04 | インディアナ ユニヴァーシティ リサーチ アンド テクノロジー コーポレイション | Incretin-insulin conjugate |
| ES2947409T3 (en) | 2014-09-24 | 2023-08-08 | Univ Indiana Res & Tech Corp | Lipid amide-based insulin prodrugs |
| EP3212218B1 (en) | 2014-10-29 | 2021-06-30 | Zealand Pharma A/S | Gip agonist compounds and methods |
| PH12017500935A1 (en) | 2014-11-21 | 2017-11-27 | Merck Sharp & Dohme | Insulin receptor partial agonists |
| EP3268384B1 (en) | 2015-03-10 | 2021-11-03 | Merck Sharp & Dohme Corp. | Process for preparing recombinant insulin using microfiltration |
| US10336802B2 (en) | 2015-04-16 | 2019-07-02 | Zealand Pharma A/S | Acylated glucagon analogue |
| UY36870A (en) | 2015-08-28 | 2017-03-31 | Hanmi Pharm Ind Co Ltd | NEW INSULIN ANALOGS |
| EP3344651B1 (en) | 2015-09-02 | 2022-03-02 | Merck Sharp & Dohme Corp. | A process for obtaining insulin with correctly formed disulfide bonds |
| US20190142963A1 (en) | 2016-04-15 | 2019-05-16 | Richard D. DiMarchi | Fgf21 c-terminal peptide optimization |
| EP3448417A4 (en) | 2016-04-26 | 2019-12-11 | Merck Sharp & Dohme Corp. | SOUND CONJUGATES OF INSULIN-INCRETIN |
| WO2017205191A1 (en) | 2016-05-24 | 2017-11-30 | Merck Sharp & Dohme Corp. | Insulin receptor partial agonists and glp-1 analogues |
| WO2017205309A1 (en) | 2016-05-25 | 2017-11-30 | Merck Sharp & Dohme Corp. | Insulin receptor partial agonists |
| EP3464336B1 (en) | 2016-06-01 | 2022-03-16 | Athira Pharma, Inc. | Compounds |
| WO2017210168A1 (en)* | 2016-06-02 | 2017-12-07 | Indiana University Research And Technology Corporation | Aqueously soluble and chemically stable depsi glucagon agonists |
| KR20190026786A (en) | 2016-07-18 | 2019-03-13 | 에테하 취리히 | β-cell-mimic cells |
| EP3272877A1 (en) | 2016-07-18 | 2018-01-24 | ETH Zurich | B-cell-mimetic cells |
| EP3517544B1 (en) | 2016-09-23 | 2025-01-08 | Hanmi Pharm. Co., Ltd. | Insulin analog having reduced binding force to insulin receptor, and use thereof |
| UY37629A (en) | 2017-03-07 | 2018-10-31 | Univ Case Western Reserve | SIMPLE CHAIN INSULIN ANALOGS STABILIZED BY A FOUR DISULFURED BRIDGE |
| AR111341A1 (en) | 2017-03-23 | 2019-07-03 | Hanmi Pharm Ind Co Ltd | AN INSULIN ANALOG WITH REDUCED AFFINITY FOR THE INSULIN RECEIVER AND USE OF IT |
| EP3600381A4 (en) | 2017-03-23 | 2021-06-16 | Merck Sharp & Dohme Corp. | GLUCOSE-ACTIVE INSULIN WITH A THREE-VALUE SUGAR CLUSTER FOR THE TREATMENT OF DIABETES |
| WO2018213151A1 (en) | 2017-05-18 | 2018-11-22 | Merck Sharp & Dohme Corp. | Pharmaceutical formulation comprising incretin-insulin conjugates |
| CN111032685A (en) | 2017-08-17 | 2020-04-17 | 诺沃挪第克公司 | Novel acylated insulin analogs and their uses |
| TW202039437A (en) | 2018-12-11 | 2020-11-01 | 法商賽諾菲公司 | Peptide binder |
| WO2020247297A1 (en) | 2019-06-06 | 2020-12-10 | Merck Sharp & Dohme Corp. | Glucose-responsive insulin conjugates |
| BR112022024058A2 (en)* | 2020-05-26 | 2022-12-20 | Univ Indiana Res & Tech Corp | PTH ANALOGS FOR THE TREATMENT OF HYPOPARATHYROIDISM |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341122A (en)* | 1999-02-24 | 2002-03-20 | 阿文蒂斯药物德国有限公司 | Covalently bridged insulin dimers |
| US20060240053A1 (en)* | 2002-07-09 | 2006-10-26 | L'oreal | Use of aspartic proteases in cosmetics and therapeutics |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3740385A (en) | 1970-05-07 | 1973-06-19 | M Ondetti | N-terminal derivatives of secretin |
| US4275152A (en) | 1977-02-03 | 1981-06-23 | Eastman Kodak Company | Hydrolysis of protein-bound cholesterol esters |
| PT83613B (en) | 1985-10-28 | 1988-11-21 | Lilly Co Eli | Process for the selective chemical removal of a protein amino-terminal residue |
| EP0272253A4 (en) | 1986-03-07 | 1990-02-05 | Massachusetts Inst Technology | Method for enhancing glycoprotein stability. |
| US4741897A (en) | 1986-07-08 | 1988-05-03 | Baxter Travenol | Thyroxine analogs and reagents for thyroid hormone assays |
| US4876242A (en) | 1987-09-21 | 1989-10-24 | Merck & Co., Inc. | Human insulin-like growth factor analoges with reduced binding to serum carrier proteins and their production in yeast |
| GB8728561D0 (en) | 1987-12-07 | 1988-01-13 | Glaxo Group Ltd | Chemical compounds |
| US5514646A (en) | 1989-02-09 | 1996-05-07 | Chance; Ronald E. | Insulin analogs modified at position 29 of the B chain |
| HU210142B (en) | 1989-04-20 | 1995-02-28 | Sinai School Medicine | Process for producing hepatospecific insulin-analogues and pharmaceutical compositions containing them |
| US5268453A (en) | 1991-08-08 | 1993-12-07 | Scios Inc. | Tissue-selective insulin analogs |
| US5359030A (en) | 1993-05-10 | 1994-10-25 | Protein Delivery, Inc. | Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same |
| US6476290B1 (en) | 1995-04-19 | 2002-11-05 | Dalhousie University | Transgenic tilapia comprising a humanized insulin gene |
| EP0741188A3 (en) | 1995-05-05 | 1999-07-14 | Eli Lilly And Company | Single chain insulin with high bioactivity |
| US6180767B1 (en) | 1996-01-11 | 2001-01-30 | Thomas Jefferson University | Peptide nucleic acid conjugates |
| EP0932614A1 (en) | 1996-09-09 | 1999-08-04 | Zealand Pharmaceuticals A/S | Peptide prodrugs containing an alpha-hydroxyacid linker |
| UA65549C2 (en) | 1996-11-05 | 2004-04-15 | Елі Ліллі Енд Компані | Use of glucagon-like peptides such as glp-1, glp-1 analog, or glp-1 derivative in methods and compositions for reducing body weight |
| NZ506447A (en) | 1998-02-23 | 2002-11-26 | Neurocrine Biosciences Inc | Peptide analogues of insulin with alanine substitutions and pharmaceutical use |
| EP1950223A3 (en) | 1998-03-09 | 2009-05-13 | Zealand Pharma A/S | Pharmacologically active peptide conjugates having a reduced tendency towards enzymatic hydrolysis |
| US6875741B2 (en) | 1998-09-02 | 2005-04-05 | Renuka Pillutla | Insulin and IGF-1 receptor agonists and antagonists |
| US20030236190A1 (en) | 1998-09-02 | 2003-12-25 | Renuka Pillutla | Isulin and IGF-1 receptor agonists and antagonists |
| AU3441400A (en) | 1999-03-29 | 2000-10-16 | Uutech Limited | Peptide |
| US6746853B1 (en) | 1999-05-19 | 2004-06-08 | Xencor, Inc. | Proteins with insulin-like activity useful in the treatment of diabetes |
| AUPQ661800A0 (en) | 2000-03-31 | 2000-05-04 | Metabolic Pharmaceuticals Limited | Insulin-potentiating compounds |
| CA2417100A1 (en) | 2000-08-02 | 2002-02-07 | Theratechnologies Inc. | Modified peptides with increased potency |
| GB2382346B (en) | 2000-08-04 | 2004-08-11 | Dmi Biosciences Inc | Method of synthesizing diketopiperazines |
| WO2002026265A2 (en) | 2000-09-29 | 2002-04-04 | Schering Corporation | Pegylated interleukin-10 |
| KR100449454B1 (en) | 2000-10-02 | 2004-09-21 | 이현철 | Vector for Curing Diabetes Mellitus Containing Gene of Single-chain Insulin Analog |
| EP1243276A1 (en) | 2001-03-23 | 2002-09-25 | Franciscus Marinus Hendrikus De Groot | Elongated and multiple spacers containing activatible prodrugs |
| JP2004537984A (en) | 2001-04-19 | 2004-12-24 | ザ スクリップス リサーチ インスティテュート | Methods and compositions for producing orthogonal tRNA-aminoacyl tRNA synthetase pairs |
| HUP0700126A2 (en)* | 2001-12-20 | 2007-06-28 | Lilly Co Eli | Insulin molecule having protracted time action |
| GB2392994A (en)* | 2002-05-30 | 2004-03-17 | Medivance Instr Ltd | Apparatus and method for monitoring the efficacy of an X-ray or photographic development process |
| JP2006524039A (en) | 2003-01-09 | 2006-10-26 | マクロジェニクス，インコーポレーテッド | Identification and production of antibody containing mutant Fc region and use thereof |
| US20050059605A1 (en) | 2003-01-31 | 2005-03-17 | Krishna Peri | Chemically modified metabolites of regulatory peptides and methods of producing and using same |
| WO2004078777A2 (en) | 2003-03-04 | 2004-09-16 | Biorexis Pharmaceutical Corporation | Dipeptidyl-peptidase protected proteins |
| KR100733940B1 (en) | 2003-03-19 | 2007-06-29 | 일라이 릴리 앤드 캄파니 | LP-1 Compound Connected with Polyethylene Glycol |
| EP1620118B1 (en) | 2003-04-08 | 2014-06-18 | Yeda Research And Development Co., Ltd. | Reversible pegylated drugs |
| TWI353991B (en) | 2003-05-06 | 2011-12-11 | Syntonix Pharmaceuticals Inc | Immunoglobulin chimeric monomer-dimer hybrids |
| BR122019021416A2 (en) | 2003-09-19 | 2019-12-21 | ||
| WO2005035003A2 (en) | 2003-09-22 | 2005-04-21 | Dihedron Corporation | Compositions and methods for increasing drug efficiency |
| SI1680443T1 (en) | 2003-11-05 | 2014-01-31 | Dana-Farber Cancer Institute, Inc. | Stabilized alpha helical peptides and uses thereof |
| AU2004295023A1 (en) | 2003-12-03 | 2005-06-16 | Novo Nordisk A/S | Single-chain insulin |
| CA2582552A1 (en) | 2004-10-21 | 2006-05-04 | Igf Oncology, Llc | Toxins and radionuclides coupled to igf-1 receptor ligands for treatment of cancer |
| US7521211B2 (en) | 2005-04-05 | 2009-04-21 | Regeneron Pharmaceuticals, Inc | IGF-1 and IGF-2 chimeric polypeptides and therapeutic uses thereof |
| US7846445B2 (en) | 2005-09-27 | 2010-12-07 | Amunix Operating, Inc. | Methods for production of unstructured recombinant polymers and uses thereof |
| WO2007096332A1 (en)* | 2006-02-21 | 2007-08-30 | Novo Nordisk A/S | Single-chain insulin analogues and pharmaceutical formulations thereof |
| EP1996224B1 (en) | 2006-03-15 | 2012-11-07 | Novo Nordisk A/S | Mixtures of amylin and insulin |
| CA2660835A1 (en) | 2006-08-17 | 2008-02-21 | Amylin Pharmaceuticals, Inc. | Dpp-iv resistant gip hybrid polypeptides with selectable propperties |
| CL2007002502A1 (en) | 2006-08-31 | 2008-05-30 | Hoffmann La Roche | VARIANTS OF THE SIMILAR GROWTH FACTOR TO HUMAN INSULIN-1 (IGF-1) PEGILATED IN LISIN; METHOD OF PRODUCTION; FUSION PROTEIN THAT UNDERSTANDS IT; AND ITS USE TO TREAT ALZHEIMER'S DISEASE. |
| CA2663083A1 (en) | 2006-09-08 | 2008-03-13 | Ambrx, Inc. | Modified human plasma polypeptide or fc scaffolds and their uses |
| EP2185701A4 (en) | 2007-08-15 | 2011-03-02 | Amunix Operating Inc | Compositions and methods for modifying properties of biologically active polypeptides |
| EP2036923A1 (en) | 2007-09-11 | 2009-03-18 | Novo Nordisk A/S | Improved derivates of amylin |
| EP2036539A1 (en) | 2007-09-11 | 2009-03-18 | Novo Nordisk A/S | Stable formulations of amylin and its analogues |
| ES2632504T3 (en) | 2007-11-20 | 2017-09-13 | Ambrx, Inc. | Modified insulin polypeptides and their uses |
| WO2009099763A1 (en) | 2008-01-30 | 2009-08-13 | Indiana University Research And Technology Corporation | Ester-based peptide prodrugs |
| EP3981761A3 (en) | 2008-02-01 | 2022-08-24 | Ascendis Pharma A/S | Intermediates for prodrugs |
| WO2010011313A2 (en) | 2008-07-23 | 2010-01-28 | President And Fellows Of Harvard College | Ligation of stapled polypeptides |
| CN102325539A (en) | 2008-12-19 | 2012-01-18 | 印第安纳大学研究及科技有限公司 | Amide-based glucagon superfamily peptide prodrugs |
| EP2376098A4 (en) | 2008-12-19 | 2014-06-11 | Univ Indiana Res & Tech Corp | MEDICINAL AGENTS RELATED BY DIPEPTIDES |
| JP2013540102A (en) | 2010-06-24 | 2013-10-31 | インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーション | Peptide prodrugs of the glucagon superfamily modified via amide bonds |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341122A (en)* | 1999-02-24 | 2002-03-20 | 阿文蒂斯药物德国有限公司 | Covalently bridged insulin dimers |
| US20060240053A1 (en)* | 2002-07-09 | 2006-10-26 | L'oreal | Use of aspartic proteases in cosmetics and therapeutics |
| Publication number | Publication date |
|---|---|
| CA2744558A1 (en) | 2010-07-15 |
| AU2009335715A1 (en) | 2010-07-15 |
| JP5755566B2 (en) | 2015-07-29 |
| US8697632B2 (en) | 2014-04-15 |
| CN102245624A (en) | 2011-11-16 |
| JP2012512901A (en) | 2012-06-07 |
| US20110257076A1 (en) | 2011-10-20 |
| AU2009335715B2 (en) | 2016-09-15 |
| EP2376521B1 (en) | 2016-04-13 |
| WO2010080609A1 (en) | 2010-07-15 |
| EP2376521A4 (en) | 2014-06-25 |
| EP2376521A1 (en) | 2011-10-19 |
| Publication | Publication Date | Title |
|---|---|---|
| CN102245624B (en) | Insulin prodrug based on amide | |
| CN103119057B (en) | Insulin prodrug based on amide | |
| JP5789515B2 (en) | Insulin analogue | |
| CA2802485C (en) | Single chain insulin agonists exhibiting high activity at the insulin receptor | |
| RU2678134C2 (en) | Insulin-incretin conjugates | |
| US20110245164A1 (en) | Yl-based insulin-like growth factors exhibiting high activity at the insulin receptor | |
| US10385107B2 (en) | Lipidated amide-based insulin prodrugs | |
| JP2015508285A (en) | CTP insulin analogue for the treatment of diabetes | |
| CA2886228A1 (en) | Insulin analog dimers | |
| WO2011163460A1 (en) | Yl-based insulin-like growth factors exhibiting high activity at the insulin receptor | |
| WO2017210077A1 (en) | Single chain insulin prodrugs |
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